Peri-event LFP rasters

1. EFFECT OF PHENCYCLIDINE ON HIPPOCAMPAL SENSORY GATING UNDER ISOFLURANE ANAESTHESIA IN THE RAT Dilshani Dissanayake, Charles Marsden and Robert Mason Institute of Neuroscience, School of Biomedical Sciences, University of Nottingham Medical School, QMC, UK. • INTRODUCTION • Sensory gatingis a mechanism which allows filtering of irrelevant sensory information, so enabling efficient information processing within the CNS. • Sensory gating can be assessed using an auditory Conditioning-Test paradigm which measures the reduction in the auditory evoked response (AER) produced by a test stimulus following an initial conditioning stimulus (Bickford-Wimer et al, 1990; Miller & Freedman, 1995) . • Schizophrenic patients demonstrate a lack of attenuation of the test response measured electrophysiologically by the P50 wave component of the cortical evoked potential. • In rats, a similar defect in the N40 wave, recorded from the CA3 region of the hippocampus, has been observed in pharmacologically-induced (e.g. amphetamine, phencyclidine) models of schizophrenia (Joy, McMahon & Sheppard, 2004). • The NMDA receptor antagonist phencyclidine (PCP) has been shown to produce schizophrenic symptoms in human subjects. • This study examined auditory gating in the rat hippocampus following a single dose of PCP (1mg/kg, i.p)using in vivo electrophysiology. • METHODS • Male Lister-hooded rats (n=11) were anaesthetised with isoflurane & N2O:O2 (50%:50%). • Stereotactically manipulated 16-channel microwire electrode arrays (2x8 array running medio-laterally; NB Labs, USA) were centred on the hippocampal CA1, CA3 regions and dentate gyrus (Paxinos & Watson, 1998). • Paired auditory stimuli (3kHz tones, intensity 90dB, duration 10ms) separated by 0.5s were binaurally presented through hollow ear bars. Stimuli were repeated for 128 trials with an inter-trial interval of 10s. • Simultaneous multiple single unit and local field potential (LFP) activity was recorded using a Plexon Multineuron Acquisition Processor (MAP) system (Plexon Inc., Texas, USA). Neural signals were split at the Plexon PBX preamplifier, amplified x1000 and filtered (LFPs: 0.7-170Hz; spikes: 500Hz-5kHz). • The effect of PCP (1mg/kg, i.p) and clozapine (5mg/kg, i.p) on sensory gating was compared to basal recording. • Data were analysed using NEX v3 (Neuroexplorer Inc.,USA) and Matlab v7.04. • Gating was assessed by measuring the ratio of the N40 LFP amplitude of the test (T) to the conditioning (C) response; a T/C ratio < 50% indicates gating was present. RESULTS FIG 1: Hippocampal auditory evoked single-unit and LFP responses FIG 3: Effect of PCP on auditory gating in CA3 and dentate gyrus A CA3 dentate gyrus LFP2 Averaged over 128 trials B Peri-event rasters Loss of sensory gating Sensory gating intact Averaged LFPS (A) Representative recording of auditory-evoked responses illustrating spike rasters of four single-units (upper panel) and local field potentials (lower panel) from dentate gyrus (LFP1) and CA3 (LFP2). (B) Averaged local field potentials recorded from CA3 for 128 stimulus presentation trials demonstrated a reduction in test (T) response amplitude compared to conditioning (C) response amplitude; CS = Conditioning stimulus, TS = Test response. Averaged LFPs and peri-event LFP rasters recorded from CA3 and dentate gyrus, basal and 45 minutes after PCP administration, demonstrating a significant PCP-induced disruption of gating in CA3. PCP failed to disrupt sensory gating in dentate gyrus. FIG 5: Auditory evoked unit responses in CA3 and dentate gyrus in gating rats FIG 4: Effect of clozapine on auditory gating in CA3 FIG 6: Auditory-evoked unit responses in CA3 and dentate gyrus following PCP B A A B Excitatory response (17/87 units) - Responses at 30-50ms and 100-150ms post-CS. - Little/no responses post-TS, indicating gating of unit activity • Inhibitory response (40/87 units) • Inhibition of unit activity during 100ms post-CS followed by responses at 100ms-200ms and 300-400ms. - No stimulus-evoked activity following TS. C No response (30/87 units) - No change in activity following either CS or TS compared to basal. Frequency Spikes/sec (A) Averaged LFPs and peri-event LFP rasters recorded from CA3 following 128 trials of auditory stimuli demonstrating the effects of clozapine (5mg/kg, i.p.). Clozapine increased the amplitudes of both conditioning and test responses with an increase in T/C ratio, and prevented the PCP-induced gating deficits. (B) A histogram summarising the effects of PCP and clozapine on auditory gating (T/C ratio) and the effect of clozapine on PCP-induced gating deficits. Representative single-unit PSTHs computed 45mins after PCP (1mg/kg, i.p.). (A) Attenuated sensory-gated unit activity following both CS and TS. (B) & (C) No activity during 100ms post-CS, with stimulus-evoked responses observed 100-200ms post-CS and 100-200ms post-TS. Inset: histological verification of hippocampal recording site(s). TS CS Representative single-unit peri-stimulus time histograms (PSTHs) during 1s pre- and post-condition stimulus period. Single-units were classified according to the type of response demonstrated during the 100ms period following the conditioning stimulus. DISCUSSION • The variability of single-unit activity may reflect differences in pyramidal and interneurone activity in response to auditory stimuli; pyramidal neurons are probably excited by the conditioning stimulus and later inhibited by interneurone activity in response to the test stimulus. • This study shows that a single administration of PCP selectively disrupts sensory gating in the CA3 region of the hippocampus in the isoflurane anesthetised rat. • Similar deficits are observed in schizophrenic patients and this method may provide an animal model with good predictive validity, a view substantiated by the fact that clozapine corrects the sensory gating deficits induced by PCP. • The dentate gyrus and CA3 demonstrated auditory gating in the LFP responses; no gating was observed in CA1. Some rats showed absence of gating in both CA3 and dentate gyrus under basal conditions. • A single dose of PCP (1mg/kg, i.p.) abolished sensory gating in the CA3, while the dentate gyruswas unaffected by PCP administration. • Single-unit activity following TS was lower compared to the activity in the post-CS period in gating rats. In PCP-treated rats, post-TS activity was higher than the post-CS period, indicating disruption of gating at the single-unit level. • Clozapine (5mg/kg, i.p.) prevented PCP-induced LFP gating deficits in CA3, consistent with the results of human studies showing the ability of clozapine to correct auditory gating deficits in schizophrenic patients. ACKNOWLEDGEMENTS The government of Sri Lanka and the International Office of the University of Nottingham for funding the study. Harvey Wiggins and team at Plexon Inc USA. REFERENCES Bickford-Wimer PC, et al (1990) Auditory sensory gating in hippocampal neurons: A model system in the rat. Biol. Psychiatry27: 183-192. Herbert, P et al (1996) Gating of auditory P50 in schizophrenia: unique effects of clozapine. Biol. Psychiatry40: 181-188. Joy B, McMahon RP & Sheppard PD (2004) Effects of acute and chronic clozapine on D-amphetamine induced disruption of auditory gating in the rat. Psychopharmacology174: 274-82 Miller CL & Freedman R (1995) The activity of hippocampal interneurons and pyramidal cells during the response of the hippocampus to repeated auditory stimuli. Neuroscience69: 371-81. Paxinos G & Watson C (1998) The Rat Brain in stereotaxic coordinates, 4th edition, Academic Press. FIG 2: Auditory-evoked LFP responses from CA1, CA3 and dentate gyrus Peri-event LFP rasters 128 Trials 1 CA1 Medial electrode Averaged LFPs No AERs Lateral electrode Dentate gyrus Sensory gating T/C = 17 ± 3% T/C = 26.6 ± 2.8% CA3 Dentate gyrus Non gating T/C = 88.3 ± 12% T/C = 98.8 ± 13% CA3 Amplitude (mv) Time (sec) Averaged LFPs with peri-event LFP rasters recorded from different hippocampal regions. Auditory-evoked responses were not detected in CA1, while dentate gyrus and CA3 demonstrated gating (n=5) and non-gating (n=3) responses.