Oscillations in the brain’s electrical potential, recorded through the technique of electroencephalography (EEG), reflect the ensemble activity of a large population of neurons. Auditory steady-state response (ASSR) is the time-locked entrainment in EEG to an auditory stimulus such as a train of clicks. ASSR to a 40 Hz (gamma frequency) click train is especially reduced in schizophrenia patients, reflecting the sensory processing deficits that impact real-world functional outcomes. Since the 40 Hz ASSR is demonstrable across species and is responsive to pharmacological treatments, it can be a translational biomarker for drug development studies. Prototypical antipsychotic drugs (APDs) like haloperidol and clozapine are examples of typical and atypical classes used to treat schizophrenia patients. While both are D2 receptor blockers, they have additional pharmacological effects that may differentiate them. Here, we investigated the acute effect of clozapine (atypical) or haloperidol (typical) on the 40 Hz ASSR, in two independent studies. The doses for the two drugs were chosen to reflect comparable in vivo D2 receptor occupancy. We used female Sprague-Dawley rats implanted with epidural EEG recording electrodes. In the first experiment, vehicle or clozapine 2.5, 5, and 10 mg/kg were administered (sc) and the 40 Hz ASSR paradigm (65 dB, 40 clicks for 1 second, 2-sec inter-stimulus interval) was used to record responses at 30, 60, 90 and 120 minutes post-drug. Resting-state EEG was recorded at 60 minutes post-treatment. Treatment effects were evaluated on the evoked power and phase-locking factor (PLF), a measure of trial-to-trial consistency of the 40 Hz ASSR. Clozapine improved both measures in a dose and time-dependent manner. Clozapine also tended to reduce the resting-state gamma (30-100 Hz) power, a hallmark of cortical noise. However, the effect was not significant (P>0.05). Next, we tested the effect of haloperidol on the 40 Hz ASSR. Doses of 0.02 mg/kg -0.08 mg/kg (sc) were evaluated at 30, 60, 90 and 120-minutes post-injection. Haloperidol failed to improve the 40 Hz ASSR (evoked power and PLF). Moreover, it had no discernible effect on the resting-state gamma. These results show that despite the comparable blockade of D2 receptors, the putative target for these APDs, clozapine, and haloperidol have different effects on the 40 Hz ASSR. We conclude that the effects of clozapine on 40 Hz ASSR may be unrelated to its affinity to D2 receptors and may be mediated through other pharmacological mechanisms.

The 40 Hz auditory steady state response (ASSR) is an EEG response of local neural synchrony that is evoked by the repeated presentation of a 40 Hz click train. While the principal cortical generators of this response appear to be the bilateral primary auditory cortices as they show the largest phase locking and evoked power, other regions across the cortical mantle synchronize too, including the prefrontal cortex (PFC) that receives input from the primary auditory cortex and is involved in higher order cognitive functions. In schizophrenia, it is hypothesized that NMDA-mediated disruption in PFC function contributes to cognitive deficits including working memory and executive function. In rodents, NMDA antagonists reliably disrupt set shifting, a working memory task linked to PFC function. It is however not known if NMDA antagonism would disrupt the 40 Hz ASSR in PFC. In the following study, we equipped a group of female SD rats with epidural electrodes targeting the PFC (2.5 mm anterior and 0.8 mm lateral to bregma) and the primary auditory cortex (4.5 mm caudal, 7.5 mm lateral and 3.5 mm ventral to bregma). Two epidural screw electrodes on cerebellum served as ground and reference. After recovery from surgery and acclimation, rats were pretreated with small to modest doses of the NMDA antagonist MK801 (0.025, 0.05 and 0.1 mpk) or saline (1 ml/kg, sc) in a cross-over design, tethered to EEG cables and the EEG signal was amplified and acquired (Signal 7.0; CED1401 Micro 3). Trains of square waves (~ 1 ms duration; 40/s) were generated and played through the house speakers at ~ 65 dB SPL. EEG was acquired as 4 s sweeps while the click train played between 1-2 s of each sweep; 75 trials were recorded from each subject. Sixty minutes after vehicle treatment, robust EEG entrainment was noted in both the temporal cortex as well as the PFC. As expected, the EEG signal power from the temporal cortex was notably larger compared to the PFC. Nevertheless, both regions showed clear 40 Hz entrainment to click trains. However, MK801 effect on the 40 Hz ASSR was disparate across the two regions. In the prefrontal cortex, the intertrial coherence (ITC) of the 40 Hz ASSR was strongly disrupted by MK801 at all doses (P<0.001; Dunnett’s test). Evoked power was significantly reduced only at the highest dose (P<0.0001). In primary auditory cortex, relative to vehicle treatment, evoked power showed a significant increase after 0.025 mpk and 0.05 mpk dose but declined significantly after the 0.1 mpk dose (P<0.05). However, ITC was unaffected (P>0.05). These results indicate that gamma neural synchrony in the PFC is more vulnerable to NMDA antagonist- mediated disruption, as compared to the primary auditory cortex. Moreover, it suggests that executive and cognitive functions may be more readily compromised by NMDA-mediated transmission disturbance even as auditory processing is enhanced or unaffected.

Schizophrenia is a troubling and severe mental illness that is only incompletely treated by currently available drugs. New drug development is hindered by a scarcity of functionally relevant pharmacodynamic biomarkers that are translatable across preclinical and human subjects. Although psychosis is a major feature of schizophrenia, cognitive and negative symptoms determine the long-term functional outcomes for patients. Stimulus-evoked neural synchrony at gamma (~ 40 Hz) frequency plays an important role in the processing and integration of sensory information. Not surprisingly, schizophrenia patients show deficits in gamma oscillations. NMDA receptor (NMDAR) activation on fast-spiking parvalbumin-positive interneurons is deemed important for the generation of gamma oscillations. NMDA hypofunction has been proposed as an alternative hypothesis to the well-known dopamine dysregulation to explain the neurochemical abnormalities associated with schizophrenia. For this dissertation, we validated a preclinical model to pharmacologically probe NMDA-mediated gamma oscillations by further characterizing the auditory-steady state response (ASSR) in female Sprague Dawley rats. The ASSR is a measure of cortical neural synchrony evoked in response to periodic auditory stimuli. ASSR at 40 Hz is consistently disrupted in patients. First, we established the reliability of click train-evoked 40 Hz ASSR and tone-evoked gamma oscillations in 6 separate sessions, spread over a 3-week period. Then we established the sensitivity of these neural synchrony measures to acute NMDAR blockade using the high affinity NMDA channel blocker MK-801, using a repeated measures design. Next, we compared the reliability and sensitivity of the 40 Hz ASSR from two distinct recording sites. Results from this study showed that as compared to vertex, temporal recording showed a greater gamma synchrony. However, the temporal recording had poor test-retest reliability and lower sensitivity to MK-801-induced disruption. Lastly, we characterized the dose-response profiles of an NMDA co-agonist D-serine, an atypical (clozapine) and a typical (haloperidol) antipsychotic, on the 40 Hz ASSR. Results from these studies showed that only clozapine was effective in robustly augmenting 40 Hz ASSR. Furthermore, only clozapine pretreatment had partial protective effect against MK-801 induced ASSR disruption. Overall, this work establishes that vertex recorded 40 Hz ASSR is a reliable neural synchrony biomarker in female SD rats that is amenable for bidirectional pharmacodynamic modulation.