Abstract

Phencyclidine (PCP) is a psychotomimetic drug that elicits schizophrenia-like symptoms in healthy persons, and administration of PCP to animals is used as a pharmacological model of schizophrenia. We recently demonstrated that systemic administration of PCP to rats produces long-lasting activation of medial prefrontal cortex (mPFC) neurons with augmentation of locomotor activity, whereas direct application of PCP to mPFC neurons has little effect on their firing activity. These findings suggest that PCP-induced activation of mPFC neurons is elicited mainly via excitatory inputs from regions outside the mPFC. In the present study, we examined effects of local application of PCP to the ventral hippocampus (vHIP) on firing activity of PFC neurons in freely moving rats. PCP locally perfused into the vHIP increased spontaneous discharges of PFC neurons during perfusion with augmentation of locomotor activity. Local application of a more selective NMDA receptor antagonist, MK801, to vHIP neurons under anesthesia increased the spontaneous firing rates of most neurons directly projecting to the mPFC, whereas local application of MK801 to mPFC neurons did not induce excitatory responses in any of those neurons. The present results indicate that tonic excitatory inputs from the vHIP to the PFC may trigger development of behavioral abnormalities.

Introduction

Phencyclidine (PCP) is a psychotomimetic drug that induces schizophrenia-like symptoms in healthy individuals (Javitt and Zukkin, 1991) and exacerbates pre-existing symptoms in patients with schizophrenia (Domino and Luby, 1981). These findings have attracted great interest in understanding the neural mechanisms by which PCP modulates behavior. PCP-treated animals are considered a useful pharmacological model of schizophrenia. Recent studies show that PCP-induced long-lasting activation of the prefrontal cortex (PFC) plays a pivotal role in the development of behavioral abnormalities (Moghaddam and Adams, 1998; Suzuki et al., 2002; Jodo et al., 2003b; Takahata and Moghaddam, 2003). Disturbances of PFC function have also been indicated as a principal pathology of schizophrenia (Winn, 1994). Our previous findings suggest that tonic activation of PFC neurons by PCP involves excitatory inputs from regions outside the PFC (Suzuki et al., 2002). The hippocampus is considered a candidate source of PCP-induced excitatory inputs to the PFC because the ventral part of the hippocampus (vHIP) has a dense glutamatergic projection to the medial PFC (Jay and Witter, 1991; Jay et al., 1992), and because disinhibitory activation of CA1 pyramidal cells can be produced by inhibition of tonic γ-aminobutyric acid (GABA) inputs with N-methyl-D-aspartate (NMDA) antagonists like PCP (Grunze et al., 1996; Greene, 2001). Disturbances of hippocampal function and abnormalities in its structure have been observed in patients with schizophrenia (Hirayasu et al., 2000; Jessen et al., 2003). In the present study, we demonstrated that the hippocampus plays a principal role in producing PCP-induced tonic activation of the PFC, which triggers development of behavioral abnormalities.

Materials and Methods

Animals

Adult male Sprague Dawley rats (300–400 g, n = 53) were used in the two experiments that comprised the present study. Animals were housed in the Fukushima Medical University Animal Facility under conditions of constant temperature and humidity, with food and water available ad libitum. All efforts were made to minimize animal suffering and minimize the number of animals used. All procedures used in this study were approved by, and conducted under control of, the Fukushima Medical University Animal Care and Use Committee.

Preparation of Freely Moving Rats and Electrophysiological Recording

Rats were surgically anesthetized with pentobarbital (50 mg/kg, i.P.) and administered atropine sulfate (0.4 mg/kg, i.P.). They were then positioned in a stereotaxic frame with blunt ear bars according to the atlas of Paxinos and Watson. Next, a 30–35 mm incision was made in the skin over the skull under sterile conditions. The wound margin was infiltrated with 2% lidocaine.

A commercial micromanipulator with a tungsten microelectrode (UNIQUE MEDICAL; 150 μm ϕ, 250 μm/turn, 80 kΩ at 1000 Hz) was used to record the single-unit activity of medial prefrontal cortex (mPFC) neurons. The recording electrode was lowered unilaterally into the left mPFC (coordinates, AP +3.0 mm, ML 0.8 mm, DV 2.3–2.5 mm), and the micromanipulator was then fixed onto the cranium using dental cement. A miniature stainless steel bolt (1.4 mm diameter) that was screwed into the skull near the insertion point of the needle electrode to contact the dura mater was used as a reference. Another two bolts were screwed into the skull for EEG recording: bolt 1, AP −2.0 mm, ML 2.0 mm; bolt 2 (reference), AP +10.0 mm, ML 1.0 mm. All lead wires from the electrodes were soldered onto a miniature connector, which was anchored onto the cranium using dental cement and skull screws (diameter, 1.4 mm). Rats were also implanted with a 25G guide cannula (AG-12; EICOM, Kyoto, Japan) at an angle of 15° to the medial to dorso-ventral axis in the vicinity of the ventral hippocampus (AP −6.5 mm, ML 4.0 mm). The tip of the guide cannula was positioned 1 mm above the desired site to minimize damage to the target, and the cannula was fitted with an obturator. Animals received antibiotics after surgery. Recording began at least 14 days after recovery from surgery.

The signal from a recording electrode was fed into a bioelectric amplifier (gain = ×54 000) with a bandpass filter of 500–10 000 Hz via a miniature preamplifier (gain = ×1, JB220J; Nihon Kohden, Tokyo, Japan) directly attached to the socket on the animal's head. All signals were digitized and stored on a personal computer hard disk for off-line analysis. Unit action potentials were separated from background noise using a laboratory interface and spike detecting software (CED1401, Spike 2 ver. 5, Cambridge Electronic Design, Cambridge, UK).

Local Administration of Drugs in Freely Moving Rats

The microdialysis probe (AI-2-12; EiCOM, Kyoto, Japan) was inserted through the guide cannula at least 4 h before the beginning of recording. The surface membrane of the probe protruded 1 mm beyond the guide cannula. After inserting the probe, animals were transferred to the test box. Probes were perfused at a rate of 4 μl/min using an infusion pump (Model-100, Neuroscience, Tokyo, Japan) and a modified artificial cerebrospinal fluid (aCSF) containing 145 mM NaCl, 2.8 mM KCl, 1.2 mM MgCl, 1.2 mM CaCl and 0.2 mM ascorbate (pH 7.0–7.1) dissolved in 2.0 mM Sorenson's phosphate buffer.

After recording unit activity for 30 min as a control while perfusing aCSF, the perfusate was replaced with aCSF containing PCP (2 mM) or fresh aCSF alone (the same composition described above) using a liquid switch (SI-60, EiCOM). PCP was infused into the vHIP for 30 min (4 μl/min), and the perfusate was then replaced with aCSF. Perfusion of aCSF then continued for 60 min.

EEG Analysis

The direct EEG signal was filtered with a band-pass width of 0.5 to 100 Hz through a bioelectric amplifier (gain = ×4900) and passed to the signal acquisition system (CED1401). The EEG was sampled every 0.01 s. The digitized EEG was analyzed by power spectral analysis using commercial signal analysis software (Spike 2). The fast Fourier transform (FFT) was performed on six consecutive 10 s samples of EEG over a recording period of 120 min. Each 10 s EEG sweep was cosine-tapered, followed by the FFT. The EEG power for six consecutive EEG samples was averaged in each frequency band to produce sequential 1 min power spectral plots for frequencies of 0–50 Hz over a recording period of 120 min. The relative EEG band-power was then calculated every 1 min (sum of power within a specific frequency band divided by the average band-power of a baseline period before drug perfusion) for delta (0.5–3.9 Hz), theta (4.0–7.9 Hz), alpha (8.0–12.9 Hz) and beta (13.0–25.0 Hz) waves.

Behavioral Activity Rating

Each rat was placed in a transparent plastic box (W, 30 cm; L, 30 cm; H, 35 cm) during recording sessions. Intersecting lines were drawn on the floor of the box. All behavioral activity was recorded by a video camcorder for 120 min. The ‘general activity score’ was defined as the number of times the muzzle crossed a line on the floor of the box. This score was considered to be an index indicating both locomotor activity and stereotypy. The score was recorded every 2 min. The recording session started at least 4 h after the animal was placed in the experimental box, to give it time to adapt to the experimental environment.

Local Application of the Selective NMDA Antagonist MK801 to vHIP Neurons under Anesthesia

All animals were first anesthetized with pentobarbital (50 mg/kg), and then mounted in a stereotaxic apparatus. Burr holes were made in the skull over the caudal hippocampus and the PFC. A bipolar stimulating electrode consisting of two insulated stainless steel wires glued together (total diameter of 400 μm with only the tips exposed) was implanted into the mPFC (AP +3.0, ML 0.8, DV 3.5) ipsilateral to the drug-injection sites. Fine incisions were made in the dura and pia mater at the level of the ventral hippocampus (vHIP) to facilitate penetration of the recording electrode attached to the ejection micropipette. The recording/injection pipette assembly was inserted into the vHIP at a 15° angle medially from the vertical plane (AP −6.3 to −7.0, ML 4.0). A glass microelectrode containing 2% Pontamine Sky Blue in 0.5 M sodium acetate was used to record the impulse activity of neurons. The recording electrode had a tip diameter of ∼2 μm, and its impedance ranged from 9 to 12 MΩ. Single neural activity was passed to a microelectrode amplifier (MEZ-7200; Nihon Kohden) and amplified with a bandpass filter of 30–10 000 Hz via a bioelectric amplifier (gain = ×30 000, AVH-11; Nihon Kohden). No recordings were made until the pentobarbital anesthesia began to wear off, as indicated by sporadic whisker movement. Urethane (0.4–0.5 g/kg, i.P.) was then administered, ∼30 min before the start of recording, to maintain a moderate but stable state of anesthesia during the entire recording period. Before each drug administration, spontaneous firing activity was recorded for at least 3 min as a control (baseline activity).

To monitor the depth of anesthesia, cortical EEG was recorded through two stainless steel bolts (diameter, 1.4 mm) that were screwed to the skull to contact the dura mater (AP −2.0, ML 1.0) contralateral to the unit-recording site and the rostral tip of the olfactory bulb (AP +10.0, ML 1.0). The latter bolt was used as a reference electrode. The EEG signal was amplified using a bioelectric amplifier with bandwidth filters of 0.5–100 Hz.

Drugs were ejected using microiontophoresis or nitrogen gas pressure. Iontophoretic application of drugs was performed using six-barreled glass micropipettes glued to the recording electrode. The tip of the recording electrode protruded ∼8 μm beyond the injection tips. The injection pipettes assembly had an overall tip diameter of ∼8 μm. Two of the six barrels were filled with 3 M NaCl to balance the current. Two other barrels were filled with 40 mM dizocilpine maleate (MK801) dissolved in 200 mM NaCl solution (pH 4.0). One barrel was filled with 50 mM NMDA in 200 mM NaCl (pH 8.1) and the remaining one was filled with physiological saline (pH 7.2). Iontophoretic currents ranged from 70 to 100 nA. MK801 was retained with 5 nA of negative current and ejected with positive current. NMDA was retained with positive current and ejected with negative current. Ejection was performed using an iontophoresis circuit with automatic current neutralization (DPI-30B; Dia Medical System, Tokyo, Japan). The duration of ejection was 60 s for MK801 and physiological saline, and 10 s for NMDA.

Pressure ejection of MK801 was performed using a glass micropipette glued to the recording electrode. The recording tip protruded ∼100 μm beyond the injection tip (<40 μm). The injection pipette was filled with MK801 (0.5 mM) dissolved in aCSF, containing 122 mM NaCl, 3.1 mM KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, 0.4 mM NaH2PO4, 25 mM NaHCo3 and 10 mM glucose. The drug solution was ejected using pulses of nitrogen gas pressure (0.7–2.1; duration, 0.2 s; PicoPump PV820, World Precision Instruments, Sarasota, FL). Injection volumes were monitored by directly measuring the meniscus movement in the injection pipette (1 mm = 60 nl). A total volume of 30 nl was infused over 60 s in each administration.

After isolating a single vHIP neuron, electrophysiological responses to mPFC stimulation were recorded to confirm monosynaptic axonal projection from the recorded neuron to the mPFC. mPFC stimulation was applied as single pulses (1.0 mA, 0.5 Hz, 0.3 ms duration, 50 times) and trains of pulses (similar pulses at 500 Hz, 2–3 pulses). Antidromic spikes in vHIP neurons driven by mPFC stimulation were characterized by their fixed latency, collision with spontaneous discharge within an appropriate time interval and their ability to follow high-frequency stimulation (200–300 Hz).

Data Analysis

To analyze the firing activity of mPFC neurons in freely moving rats, we generated peristimulus time histograms (1 min bin width) for each neuron recorded from 30 min before to 90 min after drug infusion. The baseline period was defined as the 30 min period preceding the infusion. The mean and S.D. of counts per baseline bin were determined. The onset of significant increase in firing rate was defined as the first point at which the count exceeded the mean baseline activity by 2 SD within 30 min after the beginning of infusion, and also at which the ten consecutive bins had a mean value exceeding mean baseline activity by 2 SD. A significant decrease in firing rate was defined as a period during which the mean value of at least 10 consecutive bins fell below the mean baseline activity by >2 SD within 30 min after the beginning of infusion.

We calculated the relative firing rate of each neuron, which was defined as the ratio of the firing rate in each bin to the average firing rate during the baseline period. This value was used to determine the averaged effects of local application of PCP in the vHIP on mPFC neurons with different firing levels. The relative firing rate was averaged over a 30 min period of infusion in each animal to compare between the PCP-infused and control groups.

In the microejection experiments under anesthesia, we generated peristimulus time histograms (bin width, 1 s) for each neuron recorded from 3 min before to 4 min after the start of drug injection. The baseline period was defined as the 3 min period preceding the ejection. A neuron was considered significantly responsive to the drug if it satisfied the following criteria: (i) the average count of 10 consecutive bins exceeded the mean baseline activity by >2 SD (excitatory response), or the average count of 30 bins fell below the mean baseline activity by >2 SD (inhibitory response) during the first 90 s after the start of ejection; (ii) the neuron returned to the baseline firing rate; and (iii) the response was reproduced at least twice in pressure ejection, or was not reproduced by vehicle application in iontophoresis.

General behavioral activity score was counted every 2 min. The mean and SD were calculated for a control period 30 min before the infusion, and significant activation and suppression were defined in the same manner as for the firing rate.

Results in the text and figures are presented as the mean ± SEM. In all tests, we considered a P value of <0.05 to indicate statistical significance.

Histology

Animals were deeply anesthetized with pentobarbital upon completion of the experiments. For those animals that underwent infusion of PCP in a freely moving state, the position of the recording electrode was marked by passing a positive current (30 μA, 25 s). In animals that received local injection of drugs in an anesthetized state, dye was passed from the recording barrel by a constant current source (−10 μA for 3 min) to deposit a blue spot. The animals were transcardially perfused with phosphate-buffered saline containing 5% formaldehyde. The brain was then removed from each animal and stored in a 5% formalin solution. Brains were subsequently sliced coronally into 50 μm sections using a frozen microtome. Sections were stained with neutral red to facilitate localization of marks and traces created by electrodes and dialysis probes.

Results

Local Perfusion of PCP in Freely Moving Rats

Thirty-one rats were infused with artificial cerebrospinal fluid (aCSF) only or PCP (2 mM) plus aCSF into the vHIP through a dialysis probe while recording unit activity of medial PFC (mPFC) neurons. Fifteen of these rats exhibited paroxysmal abnormal EEG activity before drug or saline perfusion, possibly due to damage to the hippocampus, which is one of the most epileptogenic areas (Racine, 1978). These 15 animals were excluded from further analyses because they exhibited epileptiform EEG during a 120 min recording period: synchronized spike waves (3–5 Hz), occurring at frequent intervals (<10 min) with a duration of at least 5 s. The following data were thus obtained from the remaining 16 rats.

Infusion of PCP into the vHIP produced a sustained increase in the spontaneous firing rate of all medial PFC neurons tested (n = 8) during infusion. No neurons exhibited a significant decrease in firing rate during drug perfusion. Averaged relative firing rates of mPFC neurons over a recording period (120 min) are presented in Figure 1C. The average normalized ratio of discharge rate to baseline activity (relative firing rate) was significantly elevated during the 30 min period of PCP infusion (2.0 ± 0.3), compared with the same duration of vehicle infusion (0.9 ± 0.0; t = 3.45, df = 7, P < 0.01). There was no significant difference in baseline firing rate between PCP-infused (2.7 ± 0.7 spikes/s) and control (3.1 ± 0.4) animals. Local infusion of PCP also augmented locomotor activity accompanied by stereotypic behavior (head swaying, sniffing, etc.), the temporal course of which was roughly parallel to that of firing activity of mPFC neurons (Fig. 1D). The average general activity score was also significantly larger during the 30 min period of PCP infusion (5.1 ± 0.9), compared with the same duration of vehicle infusion (0.9 ± 0.5; t = 4.09, df = 10, P < 0.003).

Figure 1.

Effects of locally perfused PCP in the ventral hippocampus (vHIP) on firing activity of medial prefrontal cortex (mPFC) neurons and locomotor activity in freely moving rats. (A) Location of the perfusion probes in the vHIP. Thick lines represents length of the 1 mm dialysis probes. (B) Location of recording sites in the coronal section including the mPFC. Filled circles represent sites of recording neurons. The distance of each section from the bregma is indicated in mm. (C) Averaged relative firing rates of mPFC neurons per 2 min bin among all neurons tested for PCP (n = 8) or artificial cerebrospinal fluid (aCSF, n = 8). The relative firing rate was defined as the ratio of firing rate in each bin to the average firing rate during the baseline period. (D) Averaged general behavioral activity scores across all recording sessions for PCP or aCSF perfusion. The horizontal bar represents the duration of drug application. Before and after this period (30 min), aCSF was perfused in both groups. The line on each plot denotes standard error of the mean.

Figure 1.

Effects of locally perfused PCP in the ventral hippocampus (vHIP) on firing activity of medial prefrontal cortex (mPFC) neurons and locomotor activity in freely moving rats. (A) Location of the perfusion probes in the vHIP. Thick lines represents length of the 1 mm dialysis probes. (B) Location of recording sites in the coronal section including the mPFC. Filled circles represent sites of recording neurons. The distance of each section from the bregma is indicated in mm. (C) Averaged relative firing rates of mPFC neurons per 2 min bin among all neurons tested for PCP (n = 8) or artificial cerebrospinal fluid (aCSF, n = 8). The relative firing rate was defined as the ratio of firing rate in each bin to the average firing rate during the baseline period. (D) Averaged general behavioral activity scores across all recording sessions for PCP or aCSF perfusion. The horizontal bar represents the duration of drug application. Before and after this period (30 min), aCSF was perfused in both groups. The line on each plot denotes standard error of the mean.

In order to examine whether epileptiform activity contributes to a PCP-induced increase of firing activity in mPFC neurons, the relative band power of EEG was plotted against a time axis similar to that shown in Figure 1. If propagation of epileptiform activity to distant regions contributed to the PCP-induced activation of mPFC neurons described above, a corresponding increase of EEG band-power would be expected during PCP perfusion, especially in the delta and theta bands. However, the EEG power was not elevated in any bands during PCP perfusion; rather, it appeared to be decreased in all bands (Fig. 2). The statistical analysis showed that during the drug-perfusion period (30 min), the average relative band-power was significantly decreased in the PCP-perfused group compared with the control group, independent of frequency band [F(1,14) = 14.81, P < 0.002]. This indicates desynchronization of EEG during PCP perfusion.

Figure 2.

The relative EEG band power calculated every 1 min during the recording period (120 min) for delta (0.5–3.9 Hz), theta (4.0–7.9 Hz), alpha (8.0–12.9 Hz) and beta (13.0–25.0 Hz) bands. The horizontal bar represents the duration of drug application.

Figure 2.

The relative EEG band power calculated every 1 min during the recording period (120 min) for delta (0.5–3.9 Hz), theta (4.0–7.9 Hz), alpha (8.0–12.9 Hz) and beta (13.0–25.0 Hz) bands. The horizontal bar represents the duration of drug application.

Iontophoretic Application of MK801 to vHIP Neurons under Anesthesia

To examine direct effects of a more selective NMDA antagonist, MK801, on firing activity of vHIP neurons, MK801 was locally applied to vHIP neurons under anesthesia using microiontophoresis or nitrogen gas pressure, after electrophysiologically determining whether the recorded neuron projected directly to the mPFC.

Iontophoretic application of MK801 (80–100 nA, 1 min) clearly increased spontaneous firing rate in most vHIP neurons projecting to the mPFC (6/8 cells), whereas very few neurons without antidromic activation from the mPFC (1/18) exhibited significant excitation in response to local application of MK801 in the vHIP (Fig. 3B). A few neurons exhibited marked suppression of spontaneous firing activity in response to local application of MK801, independent of whether the neurons were antidromically driven from the mPFC (1/8 in driven cells; 5/18 in non-driven cells). Most neurons not directly connected to the mPFC showed little change in firing activity after local application of MK801 (13/18, Fig. 3D). The statistical analysis revealed that the ratio of neurons excited by iontophoretic application of MK801 was significantly higher among neurons antidromically driven from the mPFC than among neurons without antidromic activation (χ2 = 10.28, P < 0.02). The average baseline firing rate before injection was 1.4 ± 0.7 spikes/s in neurons with antidromic activation from the mPFC and was 4.5 ± 1.5 in neurons without such activation. Although the baseline firing rate was noticeably lower in antidromically driven neurons than in non-driven neurons, the difference was not statistically significant (t = 1.90, df = 22.3, P < 0.08).

Figure 3.

Effects of locally applied MK801 on firing activity of vHIP neurons under anesthesia. (A) A typical excitatory response of a vHIP neuron, which was antidromically driven from the mPFC, to iontophoretically applied MK801 (40 mM). Each horizontal bar above histogram denotes the duration of drug application (1 min). Electroencephalogram (EEG) was recorded from the surface of the frontal cortex with reference to a screw electrode over the olfactory bulb. (Aa) Peristimulus time histogram (PSTH) of antidromically driven vHIP responses to single pulse stimulation of the mPFC (1 mA, 0.3 ms). A waveform drawing in the chart represent the averaged spike shape from 100 traces. (Ab) Spikes follows two-pulse stimulation (200 Hz). Stimulation was given at the point designated as arrows. Asterisks indicate antidromic spikes. (Ac) A collision of an antidromic spike (*) with a spontaneous spike (•). (B) A typical vHIP neuron without antidromic activation from the mPFC exhibits little change or a weak decrease in spontaneous discharges to locally applied MK801. NMDA was applied to verify the presence of NMDA receptors on the recorded neuron and the reliable ejection of MK801 by observing the antagonizing effect against NMDA-induced excitation. Numbers over horizontal bars represent ejection current (nA). (Ba) PSTH of non-driven vHIP response to single pulse stimulation. (C) A typical excitatory response of a vHIP neuron antidromically driven from the mPFC to locally applied MK801 (0.5 mM, 30 nl/min) ejected by nitrogen gas pressure. Horizontal bars represent the duration of drug application. (D) Proportion of ventral hippocampus (vHIP) neurons excited by locally applied MK801, of the total number of neurons recorded in two ejection experiments (iontophoresis, gas-pressure). Black bars represent vHIP neurons antidromically driven from the medial prefrontal cortex (mPFC), and hatched bars represent vHIP neurons not antidromically driven from the mPFC.

Figure 3.

Effects of locally applied MK801 on firing activity of vHIP neurons under anesthesia. (A) A typical excitatory response of a vHIP neuron, which was antidromically driven from the mPFC, to iontophoretically applied MK801 (40 mM). Each horizontal bar above histogram denotes the duration of drug application (1 min). Electroencephalogram (EEG) was recorded from the surface of the frontal cortex with reference to a screw electrode over the olfactory bulb. (Aa) Peristimulus time histogram (PSTH) of antidromically driven vHIP responses to single pulse stimulation of the mPFC (1 mA, 0.3 ms). A waveform drawing in the chart represent the averaged spike shape from 100 traces. (Ab) Spikes follows two-pulse stimulation (200 Hz). Stimulation was given at the point designated as arrows. Asterisks indicate antidromic spikes. (Ac) A collision of an antidromic spike (*) with a spontaneous spike (•). (B) A typical vHIP neuron without antidromic activation from the mPFC exhibits little change or a weak decrease in spontaneous discharges to locally applied MK801. NMDA was applied to verify the presence of NMDA receptors on the recorded neuron and the reliable ejection of MK801 by observing the antagonizing effect against NMDA-induced excitation. Numbers over horizontal bars represent ejection current (nA). (Ba) PSTH of non-driven vHIP response to single pulse stimulation. (C) A typical excitatory response of a vHIP neuron antidromically driven from the mPFC to locally applied MK801 (0.5 mM, 30 nl/min) ejected by nitrogen gas pressure. Horizontal bars represent the duration of drug application. (D) Proportion of ventral hippocampus (vHIP) neurons excited by locally applied MK801, of the total number of neurons recorded in two ejection experiments (iontophoresis, gas-pressure). Black bars represent vHIP neurons antidromically driven from the medial prefrontal cortex (mPFC), and hatched bars represent vHIP neurons not antidromically driven from the mPFC.

Local Application of MK801 by Gas Pressure to vHIP Neurons under Anesthesia

A larger amount of MK801 (0.5 mM, 20 nl/min) locally ejected by gas pressure also induced notable excitation in most vHIP neurons antidromically driven from the mPFC (8/11, Fig. 3C). Only a few non-driven neurons exhibited excitation in response to the pressure-ejected MK801 (2/21, Fig. 3D). The ratio of neurons excited by the MK801 was also significantly higher among neurons antidromically driven from the mPFC (χ2 = 10.64, P < 0.002). Pure inhibitory responses to the MK801 were not detected in antidromically driven neurons (0/11), and only a few neurons without antidromic activation from the PFC (3/21) exhibited such inhibition. The average baseline firing rate was noticeably lower in antidromically driven neurons (0.4 ± 0.1) than in non-driven neurons (1.2 ± 0.4), but this difference was not statistically significant (t = 1.98, df = 22.9, P < 0.07). The locations of recording sites in the vHIP are summarized in Figure 4.

Figure 4.

Location of recording sites in the vHIP. Filled circles denote the location at which neurons exhibited excitatory responses to iontophoretically applied MK801, filled triangles denote inhibitory responses and asterisks denote no significant changes in firing rate. Filled rectangles denote excitatory responses to locally applied MK801 ejected by gas pressure, filled inverted-triangles denote inhibitory responses and open triangles denote no significant changes. AD, neurons antidrimically driven from the mPFC; ND, neurons without antidromic activation.

Figure 4.

Location of recording sites in the vHIP. Filled circles denote the location at which neurons exhibited excitatory responses to iontophoretically applied MK801, filled triangles denote inhibitory responses and asterisks denote no significant changes in firing rate. Filled rectangles denote excitatory responses to locally applied MK801 ejected by gas pressure, filled inverted-triangles denote inhibitory responses and open triangles denote no significant changes. AD, neurons antidrimically driven from the mPFC; ND, neurons without antidromic activation.

Local Application of MK801 to mPFC Neurons under Anesthesia

Using gas pressure, MK801 (0.5 mM, 20 nl/min) was applied to mPFC neurons to examine whether locally applied MK801 can excite mPFC neurons. In contrast to the results for vHIP neurons, locally applied MK801 induced no clear excitatory responses in any of the mPFC neurons recorded (0/20, Fig. 5B,C). Significant inhibitory responses were detected in three neurons. Other mPFC neurons did not exhibit noticeable changes in firing activity in response to the local application of MK801.

Figure 5.

Effects of locally applied MK801 on firing activity of mPFC neurons under anesthesia. (A) Location of recording sites in the mPFC. Filled circles denote no significant changes in firing rate, and open circles denote suppression of discharges to locally applied MK801. (B) A typical mPFC neuron exhibits a weak decrease in spontaneous discharges in response to locally applied MK801 ejected by gas pressure. (C) Another mPFC neuron shows clear suppression of discharges in response to locally applied MK801.

Figure 5.

Effects of locally applied MK801 on firing activity of mPFC neurons under anesthesia. (A) Location of recording sites in the mPFC. Filled circles denote no significant changes in firing rate, and open circles denote suppression of discharges to locally applied MK801. (B) A typical mPFC neuron exhibits a weak decrease in spontaneous discharges in response to locally applied MK801 ejected by gas pressure. (C) Another mPFC neuron shows clear suppression of discharges in response to locally applied MK801.

Discussion

The present results clearly show that local perfusion of PCP into the vHIP produces tonic activation of mPFC neurons, and concurrently induces augmentation of spontaneous locomotor activity. These findings suggest that PCP-induced abnormal inputs from the vHIP to the mPFC contribute to the long-lasting excitation of mPFC neurons observed in systemic administration of PCP, leading to development of behavioral abnormalities. The results of local injection under anesthesia also support this hypothesis. Local application of a more selective NMDA receptor antagonist, MK801, to vHIP neurons increased the spontaneous firing rates of most neurons directly projecting to the mPFC, whereas no mPFC neurons exhibited excitatory responses to locally applied MK801. These results indicate that NMDA antagonists such as PCP and MK801 selectively excite vHIP neurons connected directly to the mPFC, although the selective mechanism involved is unknown. In vitro studies have produced findings that appear related to this selective mechanism; NMDA antagonists can reduce recurrent inhibition of projection neurons in hippocampal networks, presumably by reducing NMDA receptor-dependent excitatory drive onto inhibitory interneurons, and can induce disinhibition of pyramidal cells (Grunze et al., 1996; Greene, 2001). There may be two subtypes of NMDA receptors, one of which is more sensitive to NMDA antagonists and is primarily located on inhibitory interneurons.

The lack of activation by locally applied MK801 in the mPFC under anesthesia does not rule out the possibility that the local action of NMDA antagonists in the mPFC makes a contribution to inducing or maintaining tonic activation of mPFC neurons in unanesthetized animals. However, the anesthetic method used was the same one that, in our previous study, did not block tonic activation of mPFC neurons by systemically applied PCP (Suzuki et al., 2002) or MK801 (Jodo et al., 2003a). Although anesthetic agents strongly interact with NMDA antagonists (Balster and Wessinger, 1983), the anesthetic method used do not appear to affect severely the neural mechanism responsible for PCP-induced activation of mPFC neurons in the present study. Therefore, local action of NMDA antagonists in the mPFC seems to be less involved at least in triggering activation of mPFC neurons.

When drawing conclusions regarding the present results, it is important to consider the fact that in the present experiment, the ventral hippocampus was physically penetrated with a microdialysis probe. The hippocampus is highly susceptible to kindling of epileptiform activity (Racine, 1978). Indeed, some rats were excluded from the present final analyses because they exhibited frequent abnormal EEG during the recording period. These facts suggest the possibility that activation of mPFC neurons during local perfusion of PCP is not due to local effects of PCP on vHIP neurons, but rather is due to the spread of epileptiform activity to other distant regions. If this is the case, a corresponding increase of synchronized EEG activity should be observed during PCP perfusion, especially in the delta and theta bands. However, spectral analyses showed no such increase of synchronized activity during PCP perfusion; rather, the EEG power was decreased in all bands, suggesting reduced synchronization of EEG. Therefore, it is unlikely that the spread of epileptiform activity is a primary factor in the tonic activation of mPFC neurons observed in the present study.

If neuronal dysfunction in PCP-injected animals partly involves the same mechanism as neuronal dysfunction in schizophrenia patients, long-lasting tonic activation of the PFC may be a key factor in development of pathophysiological changes in neural circuits. In a post-mortem neuropathological study of schizophrenia patients, the density of basilar dendritic spines on deep layer 3 pyramidal neurons was significantly decreased specifically in the dorsolateral PFC (Glantz and Lewis, 2000). Dendritic spines are the principal site of excitatory inputs to pyramidal neurons, and the density of these spines is considered to reflect the number of excitatory inputs (DeFelipe and Farinas, 1992). Decreased density of dendritic spines indicates a reduction of excitatory inputs to pyramidal cells in schizophrenia patients. This suggests that such structural changes are due to the down-regulation or breaking-down of excitatory synapses caused by tonic excitatory inputs over a long period. Because the dorsolateral PFC in primates anatomically corresponds to the medial PFC in rodents (Kolb, 1990), it would be worthwhile to examine whether repeated administration of PCP can produce a similar decrease in density of dendritic spines of pyramidal cells in the mPFC of rats.

Since in this study we did not examine whether inactivation of the vHIP can abolish activation of mPFC neurons by systemically administered NMDA antagonists, we cannot conclude that excitatory excess inputs from the vHIP are the only and critical pathway through which mPFC neurons are tonically activated. Further studies are required to determine this problem. The present study, however, demonstrates that NMDA antagonists like PCP and MK801, which induce schizophrenia-like symptoms in humans, selectively activate vHIP neurons directly connected to the mPFC, and that the resulting excitatory inputs from the vHIP to the mPFC make some contribution to inducing tonic excitation of mPFC neurons, in parallel with augmentation of behavioral activity. These findings also suggest that tonic excitation of PFC neurons without epileptiform activity via the excitatory pathway from the hippocampus occurs in the early stages of schizophrenia or before development of symptoms of schizophrenia. If this hypothesis is confirmed, it will be a promising clue for the development of new strategies for diagnosis and treatment of schizophrenia.

This work was supported by a Grant-in-aid for Scientific Research (No. 11670958) from the Japan Society for the Promotion of Science to the first author, and by a grant from the Fukushima Society for the Promotion of Medicine to the second author. We thank Dainippon Pharmaceutical Corporation for their generous donation of PCP. We also wish to thank Nobuko Anzai for her excellent technical assistance.

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Author notes

1Department of Physiology, Fukushima Medical University School of Medicine, 1 Hikari-ga-oka, Fukushima 960-1295, Japan and 2Department of Neuropsychiatry, Fukushima Medical University School of Medicine, 1 Hikari-ga-oka, Fukushima 960-1295, Japan