Assessing the Cntnap2 knockout rat prepulse inhibition deficit through prepulse scaling of the baseline startle response curve.

Many neurodevelopmental disorders, including autism spectrum disorder (ASD), are associated with changes in sensory processing and sensorimotor gating. The acoustic startle response and prepulse inhibition (PPI) of startle are widely used translational measures for assessing sensory processing and sensorimotor gating, respectively. The Cntnap2 knockout (KO) rat has proven to be a valid model for ASD, displaying core symptoms, including sensory processing perturbations. Here, we used a novel method to assess startle and PPI in Cntnap2 KO rats that allows for the identification of separate scaling components: startle scaling, which is a change in startle amplitude to a given sound, and sound scaling, which reflects a change in sound processing. Cntnap2 KO rats show increased startle due to both an increased overall response (startle scaling) and a left shift of the sound/response curve (sound scaling). In the presence of a prepulse, wildtype rats show a reduction of startle due to both startle scaling and sound scaling, whereas Cntnap2 KO rats show normal startle scaling, but disrupted sound scaling, resulting in the reported PPI deficit. These results validate that startle and sound scaling by a prepulse are indeed two independent processes, with only the latter being impaired in Cntnap2 KO rats. As startle scaling is likely related to motor output and sound scaling to sound processing, this novel approach reveals additional information on the possible cause of PPI disruptions in preclinical models.

A review of the neural basis underlying the acoustic startle response with a focus on recent developments in mammals.

The startle response consists of whole-body muscle contractions, eye-blink, accelerated heart rate, and freezing in response to a strong, sudden stimulus. It is evolutionarily preserved and can be observed in any animal that can perceive sensory signals, indicating the important protective function of startle. Startle response measurements and its alterations have become a valuable tool for exploring sensorimotor processes and sensory gating, especially in the context of pathologies of psychiatric disorders. The last reviews on the neural substrates underlying acoustic startle were published around 20 years ago. Advancements in methods and techniques have since allowed new insights into acoustic startle mechanisms. This review is focused on the neural circuitry that drives the primary acoustic startle response in mammals. However, there have also been very successful efforts to identify the acoustic startle pathway in other vertebrates and invertebrates in the past decades, so at the end we briefly summarize these studies and comment on the similarities and differences between species.

Spatiotemporally dynamic electric fields for brain cancer treatment: anin vitroinvestigation.

Objective. The treatment of glioblastoma (GBM) using low intensity electric fields (∼1 V cm-1) is being investigated using multiple implanted bioelectrodes, which was termed intratumoral modulation therapy (IMT). Previous IMT studies theoretically optimized treatment parameters to maximize coverage with rotating fields, which required experimental investigation. In this study, we employed computer simulations to generate spatiotemporally dynamic electric fields, designed and purpose-built an IMT device forin vitroexperiments, and evaluated the human GBM cellular responses to these fields.Approach. After measuring the electrical conductivity of thein vitroculturing medium, we designed experiments to evaluate the efficacy of various spatiotemporally dynamic fields: (a) different rotating field magnitudes, (b) rotating versus non-rotating fields, (c) 200 kHz versus 10 kHz stimulation, and (d) constructive versus destructive interference. A custom printed circuit board (PCB) was fabricated to enable four-electrode IMT in a 24-well plate. Patient derived GBM cells were treated and analyzed for viability using bioluminescence imaging.Main results. The optimal PCB design had electrodes placed 6.3 mm from the center. Spatiotemporally dynamic IMT fields at magnitudes of 1, 1.5, and 2 V cm-1reduced GBM cell viability to 58%, 37% and 2% of sham controls respectively. Rotating versus non-rotating, and 200 kHz versus 10 kHz fields showed no statistical difference. The rotating configuration yielded a significant reduction (p< 0.01) in cell viability (47 ± 4%) compared to the voltage matched (99 ± 2%) and power matched (66 ± 3%) destructive interference cases.Significance. We found the most important factors in GBM cell susceptibility to IMT are electric field strength and homogeneity. Spatiotemporally dynamic electric fields have been evaluated in this study, where improvements to electric field coverage with lower power consumption and minimal field cancellations has been demonstrated. The impact of this optimized paradigm on cell susceptibility justifies its future use in preclinical and clinical trial investigations.

Differences in Startle and Prepulse Inhibition in Contactin-associated Protein-like 2 Knock-out Rats are Associated with Sex-specific Alterations in Brainstem Neural Activity.

The contactin-associated protein-like 2 (CNTNAP2) gene encodes for the CASPR2 protein, which plays an essential role in neurodevelopment. Mutations in CNTNAP2 are associated with neurodevelopmental disorders, including autism spectrum disorder and schizophrenia. Rats with a loss of function mutation in the Cntnap2 gene show increased acoustic startle response (ASR) and decreased prepulse inhibition (PPI). The neural basis of this altered auditory processing in Cntnap2 knock-out rats is currently unknown. Auditory brainstem recordings previously revealed no differences between the genotypes. The next step is to investigate brainstem structures outside of the primary auditory pathway that mediate ASR and PPI, which are the pontine reticular nucleus (PnC) and pedunculopontine tegmentum (PPTg), respectively. Multi-unit responses from the PnC and PPTg in vivo of the same rats revealed sex-specific effects of loss of CASPR2 expression on PnC activity, but no effects on PPTg activity. Female Cntnap2-/- rats showed considerably increased PnC firing rates compared with female wildtypes, whereas the difference between the genotypes was modest in male rats. In contrast, for both females and males we found meager differences between the genotypes for PPTg firing rates and inhibition of PnC firing rates, indicating that altered firing rates of these brainstem structures are not responsible for decreased PPI in Cntnap2-/- rats. We conclude that the auditory processing changes seen in Cntnap2-/- rats are associated with, but cannot be fully explained by, differences in PnC firing rates, and that a loss of function mutation in the Cntnap2 gene has differential effects depending on sex.

Planning system for the optimization of electric field delivery using implanted electrodes for brain tumor control.

BACKGROUND: The use of non-ionizing electric fields from low-intensity voltage sources (< 10 V) to control malignant tumor growth is showing increasing potential as a cancer treatment modality. A method of applying these low-intensity electric fields using multiple implanted electrodes within or adjacent to tumor volumes has been termed as intratumoral modulation therapy (IMT). PURPOSE: This study explores advancements in the previously established IMT optimization algorithm, and the development of a custom treatment planning system for patient-specific IMT. The practicality of the treatment planning system is demonstrated by implementing the full optimization pipeline on a brain phantom with robotic electrode implantation, postoperative imaging, and treatment stimulation. METHODS: The integrated planning pipeline in 3D Slicer begins with importing and segmenting patient magnetic resonance images (MRI) or computed tomography (CT) images. The segmentation process is manual, followed by a semi-automatic smoothing step that allows the segmented brain and tumor mesh volumes to be smoothed and simplified by applying selected filters. Electrode trajectories are planned manually on the patient MRI or CT by selecting insertion and tip coordinates for a chosen number of electrodes. The electrode tip positions and stimulation parameters (phase shift and voltage) can then be optimized with the custom semi-automatic IMT optimization algorithm where users can select the prescription electric field, voltage amplitude limit, tissue electrical properties, nearby organs at risk, optimization parameters (electrode tip location, individual contact phase shift and voltage), desired field coverage percent, and field conformity optimization. Tables of optimization results are displayed, and the resulting electric field is visualized as a field-map superimposed on the MR or CT image, with 3D renderings of the brain, tumor, and electrodes. Optimized electrode coordinates are transferred to robotic electrode implantation software to enable planning and subsequent implantation of the electrodes at the desired trajectories. RESULTS: An IMT treatment planning system was developed that incorporates patient-specific MRI or CT, segmentation, volume smoothing, electrode trajectory planning, electrode tip location and stimulation parameter optimization, and results visualization. All previous manual pipeline steps operating on diverse software platforms were coalesced into a single semi-automated 3D Slicer-based user interface. Brain phantom validation of the full system implementation was successful in preoperative planning, robotic electrode implantation, and postoperative treatment planning to adjust stimulation parameters based on actual implant locations. Voltage measurements were obtained in the brain phantom to determine the electrical parameters of the phantom and validate the simulated electric field distribution. CONCLUSIONS: A custom treatment planning and implantation system for IMT has been developed in this study and validated on a phantom brain model, providing an essential step in advancing IMT technology toward future clinical safety and efficacy investigations.

Optimization of multi-electrode implant configurations and programming for the delivery of non-ablative electric fields in intratumoral modulation therapy.

PURPOSE: Application of low intensity electric fields to interfere with tumor growth is being increasingly recognized as a promising new cancer treatment modality. Intratumoral modulation therapy (IMT) is a developing technology that uses multiple electrodes implanted within or adjacent tumor regions to deliver electric fields to treat cancer. In this study, the determination of optimal IMT parameters was cast as a mathematical optimization problem, and electrode configurations, programming, optimization, and maximum treatable tumor size were evaluated in the simplest and easiest to understand spherical tumor model. The establishment of electrode placement and programming rules to maximize electric field tumor coverage designed specifically for IMT is the first step in developing an effective IMT treatment planning system. METHODS: Finite element method electric field computer simulations for tumor models with 2 to 7 implanted electrodes were performed to quantify the electric field over time with various parameters, including number of electrodes (2 to 7), number of contacts per electrode (1 to 3), location within tumor volume, and input waveform with relative phase shift between 0 and 2π radians. Homogeneous tissue specific conductivity and dielectric values were assigned to the spherical tumor and surrounding tissue volume. In order to achieve the goal of covering the tumor volume with a uniform threshold of 1 V/cm electric field, a custom least square objective function was used to maximize the tumor volume covered by 1 V/cm time averaged field, while maximizing the electric field in voxels receiving less than this threshold. An additional term in the objective function was investigated with a weighted tissue sparing term, to minimize the field to surrounding tissues. The positions of the electrodes were also optimized to maximize target coverage with the fewest number of electrodes. The complexity of this optimization problem including its non-convexity, the presence of many local minima, and the computational load associated with these stochastic based optimizations led to the use of a custom pattern search algorithm. Optimization parameters were bounded between 0 and 2π radians for phase shift, and anywhere within the tumor volume for location. The robustness of the pattern search method was then evaluated with 50 random initial parameter values. RESULTS: The optimization algorithm was successfully implemented, and for 2 to 4 electrodes, equally spaced relative phase shifts and electrodes placed equidistant from each other was optimal. For 5 electrodes, up to 2.5 cm diameter tumors with 2.0 V, and 4.1 cm with 4.0 V could be treated with the optimal configuration of a centrally placed electrode and 4 surrounding electrodes. The use of 7 electrodes allow for 3.4 cm diameter coverage at 2.0 V and 5.5 cm at 4.0 V. The evaluation of the optimization method using 50 random initial parameter values found the method to be robust in finding the optimal solution. CONCLUSIONS: This study has established a robust optimization method for temporally optimizing electric field tumor coverage for IMT, with the adaptability to optimize a variety of parameters including geometrical and relative phase shift configurations.

Diffuse intrinsic pontine glioma cells are vulnerable to low intensity electric fields delivered by intratumoral modulation therapy.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is a high fatality pediatric brain cancer without effective treatment. The field of electrotherapeutics offers new potential for other forms of glioma but the efficacy of this strategy has not been reported for DIPG. This pilot study evaluated the susceptibility of patient-derived DIPG cells to low intensity electric fields delivered using a developing technology called intratumoral modulation therapy (IMT). METHODS: DIPG cells from autopsy specimens were treated with a custom-designed, in vitro IMT system. Computer-generated electric field simulation was performed to quantify IMT amplitude and distribution using continuous, low intensity, intermediate frequency stimulation parameters. Treatment groups included sham, IMT, temozolomide (TMZ) chemotherapy and radiation therapy (RT). The impact of single and multi-modality therapy was compared using spectrophotometric and flow cytometry viability analyses. RESULTS: DIPG cells exhibited robust, consistent susceptibility to IMT fields that significantly reduced cell viability compared to untreated control levels. The ratio of viable:non-viable DIPG cells transformed from ~ 6:1 in sham-treated to ~ 1.5:1 in IMT-treated conditions. The impact of IMT was similar to that of dual modality TMZ-RT therapy and the addition of IMT to this treatment combination dramatically reduced DIPG cell viability to ~ 20% of control values. CONCLUSIONS: This proof-of-concept study provides a novel demonstration of marked DIPG cell susceptibility to low intensity electric fields delivered using IMT. The potent impact as a monotherapy and when integrated into multi-modality treatment platforms justifies further investigations into the potential of IMT as a critically needed biomedical innovation for DIPG.

The Role of Cholinergic Midbrain Neurons in Startle and Prepulse Inhibition.

One of the two major cholinergic centers of the mammalian brain is located in the midbrain, i.e., the pedunculopontine tegmentum (PPTg) and the adjacent laterodorsal tegmentum. These cholinergic neurons have been shown to be important for e.g., arousal, reward associations, and sleep. They also have been suggested to mediate sensorimotor gating, measured as prepulse inhibition of startle (PPI). PPI disruptions are a hallmark of schizophrenia and are observed in various other psychiatric disorders, where they are associated with, and often predictive of, other cognitive symptoms. PPI has been proposed to be mediated by a short midbrain circuitry including inhibitory cholinergic projections from PPTg to the startle pathway. Although the data indicating the involvement of the PPTg is very robust, some more recent evidence challenges that there is a cholinergic contribution to PPI. We here use transient optogenetic activation of specifically the cholinergic PPTg neurons in male and female rats to address their role in startle modulation in general, and in PPI specifically. Although we could confirm the crucial role of PPTg cholinergic neurons in associative reward learning, validating our experimental approach, we found that activation of cholinergic PPTg neurons did not inhibit startle responses. In contrast, activation of cholinergic PPTg neurons enhanced startle, which is in accordance with their general role in arousal and indicate a potential involvement in sensitization of startle. We conclude that noncholinergic PPTg neurons mediate PPI in contrast to the longstanding hypothetical view that PPI is mediated by cholinergic PPTg neurons.SIGNIFICANCE STATEMENT Activation of cholinergic neurons in the midbrain has been assumed to mediate prepulse inhibition of startle (PPI), a common measure of sensorimotor gating that is disrupted in schizophrenia and other psychiatric disorders. We here revisit this long-standing hypothesis using optogenetic activation of these specific neurons combined with startle testing in rats. In contrast to the hypothetical role of these neurons in startle modulation, we show that their activation leads to an increase of baseline startle and to prepulse facilitation. This supports recent data by others that have started to cast some doubt on the cholinergic hypothesis of PPI, and calls for a revision of the theoretical construct of PPI mechanisms.

Preclinical outcomes of Intratumoral Modulation Therapy for glioblastoma.

Glioblastoma (GBM) is the leading cause of high fatality cancer arising within the adult brain. Electrotherapeutic approaches offer new promise for GBM treatment by exploiting innate vulnerabilities of cancer cells to low intensity electric fields. This report describes the preclinical outcomes of a novel electrotherapeutic strategy called Intratumoral Modulation Therapy (IMT) that uses an implanted stimulation system to deliver sustained, titratable, low intensity electric fields directly across GBM-affected brain regions. This pilot technology was applied to in vitro and animal models demonstrating significant and marked reduction in tumor cell viability and a cumulative impact of concurrent IMT and chemotherapy in GBM. No off target neurological effects were observed in treated subjects. Computational modeling predicted IMT field optimization as a means to further bolster treatment efficacy. This sentinel study provides new support for defining the potential of IMT strategies as part of a more effective multimodality treatment platform for GBM.

BK Channels Mediate Synaptic Plasticity Underlying Habituation in Rats.

Habituation is a basic form of implicit learning and represents a sensory filter that is disrupted in autism, schizophrenia, and several other mental disorders. Despite extensive research in the past decades on habituation of startle and other escape responses, the underlying neural mechanisms are still not fully understood. There is evidence from previous studies indicating that BK channels might play a critical role in habituation. We here used a wide array of approaches to test this hypothesis. We show that BK channel activation and subsequent phosphorylation of these channels are essential for synaptic depression presumably underlying startle habituation in rats, using patch-clamp recordings and voltage-sensitive dye imaging in slices. Furthermore, positive modulation of BK channels in vivo can enhance short-term habituation. Although results using different approaches do not always perfectly align, together they provide convincing evidence for a crucial role of BK channel phosphorylation in synaptic depression underlying short-term habituation of startle. We also show that this mechanism can be targeted to enhance short-term habituation and therefore to potentially ameliorate sensory filtering deficits associated with psychiatric disorders.SIGNIFICANCE STATEMENT Short-term habituation is the most fundamental form of implicit learning. Habituation also represents a filter for inundating sensory information, which is disrupted in autism, schizophrenia, and other psychiatric disorders. Habituation has been studied in different organisms and behavioral models and is thought to be caused by synaptic depression in respective pathways. The underlying molecular mechanisms, however, are poorly understood. We here identify, for the first time, a BK channel-dependent molecular synaptic mechanism leading to synaptic depression that is crucial for habituation, and we discuss the significance of our findings for potential treatments enhancing habituation.

In Vitro Validation of Intratumoral Modulation Therapy for Glioblastoma.

BACKGROUND/AIM: This proof-of-concept study evaluated the antitumor impact of a direct electrical stimulation technique, termed intratumoral modulation therapy (IMT) on glioblastoma (GBM) cells. MATERIALS AND METHODS: An in vitro IMT model comprised of a calibrated electrode to deliver continuous, low-intensity stimulation within GBM preparations. Viability and apoptosis assays were performed in treated immortalized and patient-derived GBM cells, and post-mitotic neurons. IMT was delivered alone and with temozolomide, or gene silencing of the tumor-promoting chaperone, heat-shock protein 27 (HSP27). RESULTS: GBM cells, but not neurons, exhibited >40% loss of viability, caspase-3 activation and apoptosis with IMT. Cell death was modest with temozolomide alone (30%) but increased significantly with concomitant IMT (70%). HSP27 silencing alone produced 30% viability loss, with significant enhancement of target knockdown and GBM cell death (65%), when combined with IMT. CONCLUSION: These findings warrant further evaluation of IMT as a potential novel therapeutic strategy for GBM.

Glycine inhibits startle-mediating neurons in the caudal pontine reticular formation but is not involved in synaptic depression underlying short-term habituation of startle.

The mammalian startle response is controlled by glycine inhibition in the spinal cord. Evidence for additional glycine inhibition on the level of the brainstem, namely in the caudal pontine reticular nucleus (PnC), is controversial. Startle mediating PnC neurons receive fast input from sensory pathways and project to cranial and spinal motoneurons. Synaptic depression in the sensory synapses in the PnC has been indicated as underlying mechanism of short-term habituation of startle. We here performed patch-clamp recordings of PnC giant neurons in rat brain slices to test the hypothesis that the activation of glycine receptors inhibits PnC neurons and that this inhibition is involved in synaptic depression in the PnC. Glycine strongly inhibited PnC neuron activity and synaptic signalling, indicating that functional glycine receptors mediate a powerful inhibition of PnC neurons over a wide range of glycine concentrations. Strychnine reversed all glycine effects, but had no effect on PnC neurons itself. Thus, we found no evidence for a tonic glycine inhibition or for glycine activation within the primary startle pathway indicating that baseline startle reactions are unlikely to be controlled by glycine in the PnC. Most importantly, synaptic depression underlying short-term habituation was not affected by glycine or strychnine.

GABA receptors and prepulse inhibition of acoustic startle in mice and rats.

The acoustic startle reflex is strongly inhibited by a moderate-intensity acoustic stimulus that precedes the startling stimulus by roughly 10-1000 ms (prepulse inhibition, PPI). At long interstimulus intervals (ISIs) of 100-1000 ms, PPI in rats is reduced by the muscarinic receptor antagonist scopolamine. Here, we studied the role of GABA receptors in PPI at full ISI ranges in both mice and rats. In B6 mice, PPI begins and ends at shorter ISIs (4 and 1000 ms, respectively) than in Wistar rats (8 and 5000 ms). The GABA(A) antagonist bicuculline (1 mg/kg i.p.) reduced PPI at ISIs near the peak of PPI in both rats and mice. The GABA(B) antagonist phaclofen (10 or 30 mg/kg i.p. in rats or mice, respectively) reduced PPI only at long ISIs, similar to the effects of the muscarinic antagonist scopolamine (1 mg/kg i.p.). The effects of phaclofen and scopolamine were additive in rats, suggesting independent effects of GABA(B) and muscarinic receptors. Patch-clamp recordings of startle-mediating PnC (nucleus reticularis pontis caudalis) giant neurons in rat slices show that EPSCs evoked by either trigeminal or auditory fiber stimulation were inhibited by the GABA(A/C) agonist muscimol or the GABA(B) agonist baclofen via postsynaptic mechanisms. Hyperpolarization of PnC neurons by muscimol was reversed with bicuculline, indicating that postsynaptic GABA(A) receptors strongly inhibit PnC giant neurons needed for startle. Therefore, GABA receptors on PnC giant neurons mediate a substantial part of PPI, with GABA(A) receptors contributing at the peak of PPI, and GABA(B) receptors adding to muscarinic effects on PPI at long ISIs.

Synaptic depression and short-term habituation are located in the sensory part of the mammalian startle pathway.

BACKGROUND: Short-term habituation of the startle response represents an elementary form of learning in mammals. The underlying mechanism is located within the primary startle pathway, presumably at sensory synapses on giant neurons in the caudal pontine reticular nucleus (PnC). Short trains of action potentials in sensory afferent fibers induce depression of synaptic responses in PnC giant neurons, a phenomenon that has been proposed to be the cellular correlate for short-term habituation. We address here the question whether both this synaptic depression and the short-term habituation of the startle response are localized at the presynaptic terminals of sensory afferents. If this is confirmed, it would imply that these processes take place prior to multimodal signal integration, rather than occurring at postsynaptic sites on PnC giant neurons that directly drive motor neurons. RESULTS: Patch-clamp recordings in vitro were combined with behavioral experiments; synaptic depression was specific for the input pathway stimulated and did not affect signals elicited by other sensory afferents. Concordant with this, short-term habituation of the acoustic startle response in behavioral experiments did not influence tactile startle response amplitudes and vice versa. Further electrophysiological analysis showed that the passive properties of the postsynaptic neuron were unchanged but revealed some alterations in short-term plasticity during depression. Moreover, depression was induced only by trains of presynaptic action potentials and not by single pulses. There was no evidence for transmitter receptor desensitization. In summary, the data indicates that the synaptic depression mechanism is located presynaptically. CONCLUSION: Our electrophysiological and behavioral data strongly indicate that synaptic depression in the PnC as well as short-term habituation are located in the sensory part of the startle pathway, namely at the axon terminals of sensory afferents in the PnC. Our results further corroborate the link between synaptic depression and short-term habituation of the startle response.

Cellular mechanisms of the trigeminally evoked startle response.

The startle response is an important mammalian model for studying the cellular mechanisms of emotions and of learning. It consists of contractions of facial and skeletal muscles in response to sudden acoustic, tactile or vestibular stimuli. Whereas the acoustic startle pathway is well described, only a few recent studies have investigated the tactile startle pathway. It was proposed that there is a direct projection from the principal sensory nucleus to the central sensorimotor interface of the startle response, which is formed by the giant neurons in the caudal pontine reticular formation. We explored this projection in greater detail in vitro. Anterograde tracing in rat brain slices confirmed projections with large axon terminals from the ventral part of the principal sensory nucleus to the lateral caudal pontine reticular formation. Electrophysiological studies revealed a monosynaptic glutamatergic connection between principal sensory nucleus neurons and caudal pontine reticular formation giant neurons. The synapses displayed paired-pulse facilitation at high-frequency stimulation, and homosynaptic depression at 1 Hz stimulation. The latter form of plasticity is thought to underlie habituation of the startle response. Furthermore, postsynaptic currents in caudal pontine reticular formation giant neurons evoked by principal sensory nucleus neuron stimulation summed in a linear way with signals evoked by stimulation of auditory afferents. Synaptic plasticity and summation of synaptic currents correspond well with in vivo data previously published by other groups. We thus presume that these synapses mediate trigeminal input to the startle pathway.

Synaptic plasticity in the acoustic startle pathway: the neuronal basis for short-term habituation?

The aim of the present study was to analyse the cellular mechanism underlying short-term habituation of the acoustic startle response (ASR). We explored distinct synapses of the neuronal startle pathway in rat brain slices by patch-clamp recordings of giant neurons in the caudal pontine reticular formation. Presynaptic stimulation of auditory afferents by repeated bursts at 0.1 and 1 Hz led to an exponential decay of EPSC magnitudes. This homosynaptic depression (HSD) was reversible and repeatedly inducible after recovery. Many parameters of HSD in vitro match those of ASR habituation in vivo. The mechanisms underlying HSD are distinct from classical short-term plasticity: paired-pulse as well as paired-burst stimulation revealed a facilitation of the second EPSC, occurring in a much smaller time window up to interstimulus intervals of 200 ms. Pharmacological experiments demonstrated that HSD could be completely blocked by the group II and III metabotropic glutamate receptor antagonist MPPG. Similar results were obtained by CPPG, another group II and III antagonist. In contrast, HSD was not affected by the group I and II antagonist MCPG. We conclude that we found a form of synaptic depression in synapses within the primary startle pathway which correlates in many respects with short-term habituation of the ASR and which is presumably mediated by group III metabotropic glutamate receptors.