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Mutations in theSCN1Bgene have been linked to severe developmental epileptic encephalopathies including Dravet syndrome.Scn1bknockout (KO) mice modelSCN1Bloss of function disorders, demonstrating seizures, developmental delays, and early death.SCN1Bencodes the protein β1, an ion channel auxiliary subunit that also has roles in cell adhesion, neurite outgrowth, and gene expression. The goal of this project is to better understand of how loss of β1 alters information processing in the brain, resulting in seizures and associated cognitive dysfunction. Using slice electrophysiology in the CA1 region of the hippocampus from male and femaleScn1bKO mice andwild-type (WT) littermates, we found that processing of physiologically relevant patternedSchaffercollateral (SC) stimulation produces larger, prolonged depolarizations and increased spiking in KO neurons compared to WTs. KO neurons exhibit enhanced intrinsic excitability, firing more action potentials with current injection. Interestingly, SC stimulation produces smaller, more facilitating excitatory and inhibitory postsynaptic currents in KO pyramidal neurons, but larger postsynaptic potentials with the same stimulation. We also found reduced intrinsic firing of parvalbumin-expressing interneurons and disrupted recruitment of both parvalbumin- and somatostatin-expressing interneurons in response to patterned synaptic stimulation. Neuronal information processing relies on the interplay between synaptic properties, intrinsic properties that amplify or suppress incoming synaptic signals, and firing properties that produce cellular output. We found changes at each of these levels inScn1bKO pyramidal neurons, resulting in fundamentally altered information processing in the hippocampus that likely contributes to the complex phenotypes ofSCN1B-linked epileptic encephalopathies.Significance statementGenetic developmental epileptic encephalopathies have limited treatment options, in part due to our lack of understanding of how genetic changes result in dysfunction at the cellular and circuit levels.SCN1Bis a gene linked to Dravet syndrome and other epileptic encephalopathies, andScn1bknockout mice phenocopy the human disease, allowing us to study underlying neurophysiological changes. Here we found changes at all levels of neuronal information processing in brains lacking β1, including intrinsic excitability, synaptic properties, and synaptic integration, resulting in greatly enhanced input/output functions of the hippocampus. Our study shows that loss of β1 results in a complex array of cellular and network changes that fundamentally alters information processing in the hippocampus.

Mutations of

Objective A significant proportion of the more than 50 million people world-wide currently suffering with epilepsy are resistant to antiepileptic drugs (AEDs). As an alternative to AEDs, novel therapies based on cell transplantation offer an opportunity for long-lasting modification of epileptic circuits. To develop such a treatment requires careful preclinical studies in a chronic epilepsy model featuring unprovoked seizures, hippocampal histopathology, and behavioral comorbidities. Methods Transplantation of progenitor cells from embryonic medial or caudal ganglionic eminence (MGE, CGE) were made in a well-characterized mouse model of status epilepticus-induced epilepsy (systemic pilocarpine). Behavioral testing (handling and open field), continuous video-electroencephalographic (vEEG) monitoring and slice electrophysiology outcomes were obtained up to 270 days after transplantation (DAT). Post hoc immunohistochemistry was used to confirm cell identity. Results MGE progenitors transplanted into the hippocampus of epileptic mice rescued handling and open field deficits starting at 60 DAT. In these same mice, an 84-88% reduction in seizure activity was observed between 180 and 210 DAT. Inhibitory postsynaptic current frequency, measured on pyramidal neurons in acute hippocampal slices at 270 DAT, was reduced in epileptic mice but restored to naive levels in epileptic mice receiving MGE transplants. No reduction in seizure activity was observed in epileptic mice receiving intra-hippocampal CGE progenitors. Interpretation Our findings demonstrate that transplanted MGE progenitors enhance functional GABA-mediated inhibition, reduce spontaneous seizure frequency and rescue behavioral deficits in a chronic epileptic animal model more than six months after treatment. This article is protected by copyright. All rights reserved.

The tragedy of epilepsy emerges from the combination of its high prevalence, impact upon sufferers and their families, and unpredictability. Childhood epilepsies are frequently severe, presenting in infancy with pharmaco-resistant seizures; are often accompanied by debilitating neuropsychiatric and systemic comorbidities; and carry a grave risk of mortality. Here, we review the most current basic science and translational research findings on several of the most catastrophic forms of pediatric epilepsy. We focus largely on genetic epilepsies and the research that is discovering the mechanisms linking disease genes to epilepsy syndromes. We also describe the strides made toward developing novel pharmacological and interventional treatment strategies to treat these disorders. The research reviewed provides hope for a complete understanding of, and eventual cure for, these childhood epilepsy syndromes.

Interneuron-based cell transplantation is a powerful method to modify network function in a variety of neurological disorders, including epilepsy. Whether new interneurons integrate into native neural networks in a subtype-specific manner is not well understood, and the therapeutic mechanisms underlying interneuron-based cell therapy, including the role of synaptic inhibition, are debated. In this study, we tested subtype-specific integration of transplanted interneurons using acute cortical brain slices and visualized patch-clamp recordings to measure excitatory synaptic inputs, intrinsic properties, and inhibitory synaptic outputs. Fluorescently labeled progenitor cells from the embryonic medial ganglionic eminence (MGE) were used for transplantation. At 5 wk after transplantation, MGE-derived parvalbumin-positive (PV+) interneurons received excitatory synaptic inputs, exhibited mature interneuron firing properties, and made functional synaptic inhibitory connections to native pyramidal cells that were comparable to those of native PV+ interneurons. These findings demonstrate that MGE-derived PV+ interneurons functionally integrate into subtype-appropriate physiological niches within host networks following transplantation.

The postnatal functions of the Dlx1&2 transcription factors in cortical interneurons (CINs) are unknown. Here, using conditional Dlx1, Dlx2, and Dlx1&2 knockouts (CKOs), we defined their roles in specific CINs. The CKOs had dendritic, synaptic, and survival defects, affecting even PV+ CINs. We provide evidence that DLX2 directly drives Gad1, Gad2, and Vgat expression, and show that mutants had reduced mIPSC amplitude. In addition, the mutants formed fewer GABAergic synapses on excitatory neurons and had reduced mIPSC frequency. Furthermore, Dlx1/2 CKO had hypoplastic dendrites, fewer excitatory synapses, and reduced excitatory input. We provide evidence that some of these phenotypes were due to reduced expression of GRIN2B (a subunit of the NMDA receptor), a high confidence Autism gene. Thus, Dlx1&2 coordinate key components of CIN postnatal development by promoting their excitability, inhibitory output, and survival.

A significant proportion of the more than 50 million people worldwide currently suffering with epilepsy are resistant to antiepileptic drugs (AEDs). As an alternative to AEDs, novel therapies based on cell transplantation offer an opportunity for long-lasting modification of epileptic circuits. To develop such a treatment requires careful preclinical studies in a chronic epilepsy model featuring unprovoked seizures, hippocampal histopathology, and behavioral comorbidities.Transplantation of progenitor cells from embryonic medial or caudal ganglionic eminence (MGE, CGE) were made in a well-characterized mouse model of status epilepticus-induced epilepsy (systemic pilocarpine). Behavioral testing (handling and open field), continuous video-electroencephalographic (vEEG) monitoring, and slice electrophysiology outcomes were obtained up to 270 days after transplantation (DAT). Post-hoc immunohistochemistry was used to confirm cell identity.MGE progenitors transplanted into the hippocampus of epileptic mice rescued handling and open field deficits starting at 60 DAT. In these same mice, an 84% to 88% reduction in seizure activity was observed between 180 and 210 DAT. Inhibitory postsynaptic current frequency, measured on pyramidal neurons in acute hippocampal slices at 270 DAT, was reduced in epileptic mice but restored to naïve levels in epileptic mice receiving MGE transplants. No reduction in seizure activity was observed in epileptic mice receiving intrahippocampal CGE progenitors.Our findings demonstrate that transplanted MGE progenitors enhance functional GABA-mediated inhibition, reduce spontaneous seizure frequency, and rescue behavioral deficits in a chronic epileptic animal model more than 6 months after treatment. Ann Neurol 2017;82:530-542.

Abnormal GABAergic interneuron density, and imbalance of excitatory versus inhibitory tone, is thought to result in epilepsy, neurodevelopmental disorders, and psychiatric disease. Recent studies indicate that interneuron cortical density is determined primarily by the size of the precursor pool inthe embryonic telencephalon. However, factors essential for regulating interneuron allocation from telencephalic multipotent precursors are poorly understood. Here we report that Olig1 represses production of GABAergic interneurons throughout the mouse brain. Olig1 deletion in mutant mice results in ectopic expression and upregulation of Dlx1/2 genes in the ventral medial ganglionic eminences and adjacent regions of the septum, resulting in an ~30% increase in adult cortical interneuron numbers. We show that Olig1 directly represses the. Dlx1/2 I12b intergenic enhancer and that Dlx1/2 functions genetically downstream of Olig1. These findings establish Olig1 as an essential repressor of. Dlx1/2 and interneuron production in developing mammalian brain. © 2014 Elsevier Inc.

Significance We describe homeostatic plasticity of both excitatory synaptic transmission and intrinsic properties of CA1 pyramidal neurons in distal-less homeobox 1 ( Dlx1 −/− ) mice, a genetic model of postdevelopment interneuron cell death. Loss of synaptic inhibition and compensation by excitation led to enhanced potential for long-term potentiation (LTP) and altered neural oscillations. This shows that homeostatic compensation may rebalance inhibition and excitation but cannot fully normalize neural function. Transplantation of interneuron progenitor cells restored inhibitory synaptic transmission to WT levels and induced a reversal of homeostatic changes to excitation and excitability. LTP and gamma oscillations were also normalized after integration of transplanted interneurons. These data indicate that homeostatic plasticity functions in vivo to balance activity based on inhibitory tone.

Dlx transcription factors are important in the differentiation of GABAergic interneurons. In mice lacking Dlx1, early steps in interneuron development appear normal. Beginning at ∼1 mo of age, primarily dendrite-innervating interneuron subtypes begin to undergo apoptosis in Dlx1−/− mice; this is accompanied by a reduction in GABAergic transmission and late-onset epilepsy. The reported reduction of synaptic inhibition is greater than might be expected given that interneuron loss is relatively modest in Dlx1−/− mice. Here we report that voltage-clamp recordings of CA1 interneurons in hippocampal slices prepared from Dlx1−/− animals older than postnatal day 30 (>P30) revealed a significant reduction in excitatory postsynaptic current (EPSC) amplitude. No changes in EPSCs onto interneurons were observed in cells recorded from younger animals (P9–12). Current-clamp recordings from interneurons at these early postnatal ages showed that interneurons in Dlx1−/− mutants were immature and more excitable, although membrane properties normalized by P30. Terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, caspase-3, and NeuN staining did not reveal frank cell damage or loss in area CA3 of hippocampal sections from adult Dlx1−/− mice. Delayed interneuron maturation may lead to interneuron hyperexcitability, followed by a compensatory reduction in the strength of excitatory transmission onto interneurons. This reduced excitation onto surviving interneurons, coupled with the loss of a significant fraction of GABAergic inputs to excitatory neurons starting at P30, may underlie cortical dysrhythmia and seizures previously observed in adult Dlx1−/− mice.

Mature nucleus magnocellularis (NM) neurons, the avian homolog of bushy cells of the mammalian anteroventral cochlear nucleus, maintain high [Cl−]iand depolarize in response to GABA. Depolarizing GABAergic postsynaptic potentials (GPSPs) activate both the synaptic conductance and large outward currents, which, when coupled together, inhibit spikes via shunting and spike threshold accommodation. We studied the maturation of the synaptic and voltage-dependent components of inhibition in embryonic NM neurons using whole-cell and gramicidin-perforated patch-clamp techniques to measure Cl−reversal potential, GABAergic synaptic responses, and voltage-dependent outward currents. We found that GABA enhanced excitability in immature NM neurons, undergoing a switch to inhibitory between embryonic day 14 (E14) and E18. Low-voltage-activated Kv1-type (dendrotoxin-I sensitive) K+currents increased in amplitude between E14 and E18, whereas Cl−reversal potential and synaptic conductances remained relatively stable during this period. GABA was rendered inhibitory because of this increase in low-voltage activated outward currents. GPSPs summed with other inputs to increase spike probability at E14. GPSPs shunted spikes at E18, but blocking Kv1 channels transformed this inhibition to excitation, similar to E14 neurons. Subthreshold depolarizing current steps, designed to activate outward currents similar to depolarizing GPSPs, enhanced excitability at E14 but inhibited spiking in E18 neurons. Blocking Kv1 channels reversed this effect, rendering current steps excitatory. We present the novel finding that the developmental transition of GABAergic processing from increasing neuronal excitability to inhibiting spiking can depend on changes in the expression of voltage-gated channels rather than on a change in Cl−reversal potential.

Object. Although the benefits of posttraumatic hypothermia have been reported in experimental studies, the potential for therapeutic hypothermia to increase intracerebral hemorrhage remains a clinical concern. The purpose of this study was to quantify the amount of extravasated hemoglobin after traumatic brain injury (TBI) and to assess the changes in intracerebral hemoglobin concentrations under posttraumatic hypothermic and hyperthermic conditions. Methods. Intubated and anesthetized rats were subjected to fluid-percussion injury (FPI). In the first experiment, rats were divided into moderate (1.8–2.2 atm) and severe (2.4–2.7 atm) TBI groups. In the second experiment, the effects of 3 hours of posttraumatic hypothermia (33 or 30°C), hyperthermia (39°C), or normothermia (37°C) on hemoglobin levels following moderate trauma were assessed. The rats were perfused with saline at 24 hours postinjury, and then the traumatized and contralateral hemispheres, including the cerebellum, were dissected from whole brain. The hemoglobin level in each brain was quantified using a spectrophotometric hemoglobin assay. The results of these assays indicate that moderate and severe FPI induce increased levels of hemoglobin in the ipsilateral hemisphere (p < 0.0001). After severe TBI, the hemoglobin concentration was also significantly increased in the contralateral hemisphere (p < 0.05) and cerebellum (p < 0.005). Posttraumatic hypothermia (30°C) attenuated hemoglobin levels (p < 0.005) in the ipsilateral hemisphere, whereas hyperthermia had a marked adverse effect on the hemoglobin concentration in the contralateral hemisphere (p < 0.05) and cerebellum (p < 0.005). Conclusions. Injury severity is an important determinant of the degree of hemoglobin extravasation after TBI. Posttraumatic hypothermia reduced hemoglobin extravasation, whereas hyperthermia increased hemoglobin levels compared with normothermia. These findings are consistent with previous data reporting that posttraumatic temperature manipulations alter the cerebrovascular and inflammatory consequences of TBI.

Objective: Myoclonus is characterized by sudden, brief involuntary movements, and its presence is debilitating. We identified a family suffering from adult onset, cortical myoclonus without associated seizures. We performed clinical, electrophysiological, and genetic studies to define this phenotype. Methods: A large, 4-generation family with a history of myoclonus underwent careful questioning, examination, and electrophysiological testing. Thirty-five family members donated blood samples for genetic analysis, which included single nucleotide polymorphism mapping, microsatellite linkage, targeted massively parallel sequencing, and Sanger sequencing. In silico and in vitro experiments were performed to investigate functional significance of the mutation. Results: We identified 11 members of a Canadian Mennonite family suffering from adult onset, slowly progressive, disabling, multifocal myoclonus. Somatosensory evoked potentials indicated a cortical origin of the myoclonus. There were no associated seizures. Some severely affected individuals developed signs of progressive cerebellar ataxia of variable severity late in the course of their illness. The phenotype was inherited in an autosomal dominant fashion. We demonstrated linkage to chromosome 16q21-22.1. We then sequenced all coding sequence in the critical region, identifying only a single cosegregating, novel, nonsynonymous mutation, which resides in the gene NOL3. Furthermore, this mutation was found to alter post-translational modification of NOL3 protein in vitro. Interpretation: We propose that familial cortical myoclonus is a novel movement disorder that may be caused by mutation in NOL3. Further investigation of the role of NOL3 in neuronal physiology may shed light on neuronal membrane hyperexcitability and pathophysiology of myoclonus and related disorders. ANN NEUROL 2012;72:175–183.

Neurons of the cochlear nuclei are anatomically and physiologically specialized to optimally encode temporal and spectral information about sound stimuli, in part for binaural auditory processing. The avian cochlear nucleus magnocellularis (NM) integrates excitatory eighth nerve inputs and depolarizing GABAergic inhibition such that temporal fidelity is enhanced across the synapse. The biophysical mechanisms of this depolarizing inhibition, and its role in temporal processing, are not fully understood. We used whole-cell electrophysiology and computational modeling to examine how subthreshold excitatory inputs are integrated and how depolarizing IPSPs affect spike thresholds and synaptic integration by chick NM neurons. We found that both depolarizing inhibition and subthreshold excitatory inputs cause voltage threshold accommodation, nonlinear temporal summation, and shunting. Inhibition caused such large changes in threshold that subthreshold excitatory inputs were followed by a refractory period. We hypothesize that these large shifts in threshold eliminate spikes to asynchronous inputs, providing a mechanism for the enhanced temporal fidelity seen across the eighth nerve/cochlear nucleus synapse. Thus, depolarizing inhibition and threshold shifting hone the temporal response properties of this system so as to enhance the temporal fidelity that is essential for auditory perception.

Proteins of the PSD-95–like membrane-associated guanylate kinase (PSD-MAGUK) family are vital for trafficking AMPA receptors (AMPARs) to synapses, a process necessary for both basal synaptic transmission and forms of synaptic plasticity. Synapse-associated protein 97 (SAP97) exhibits protein interactions, such as direct interaction with the GluA1 AMPAR subunit, and subcellular localization (synaptic, perisynaptic, and dendritic) unique within this protein family. Due in part to the lethality of the germline knockout of SAP97, this protein’s role in synaptic transmission and plasticity is poorly understood. We found that overexpression of SAP97 during early development traffics AMPARs and NMDA receptors (NMDARs) to synapses, and that SAP97 rescues the deficits in AMPAR currents normally seen in PSD-93/-95 double-knockout neurons. Mature neurons that have experienced the overexpression of SAP97 throughout development exhibit enhanced AMPAR and NMDAR currents, as well as faster NMDAR current decay kinetics. In loss-of-function experiments using conditional SAP97 gene deletion, we recorded no deficits in glutamatergic transmission or long-term potentiation. These results support the hypothesis that SAP97 is part of the machinery that traffics glutamate receptors and compensates for other PSD-MAGUKs in knockout mouse models. However, due to functional redundancy, other PSD-MAGUKs can presumably compensate when SAP97 is conditionally deleted during development.

Cochlear function was evaluated in a longitudinal study of 28 inbred strains of mice at 3 and 5 mo of age using measures of distortion product otoacoustic emissions (DPOAEs) in response to a federal initiative to develop rapid mouse phenotyping methodologies. DP-grams at f(2) frequencies ranging from 6.3 to 54.2kHz were obtained in about 3min/ear by eliciting 2f(1)-f(2) DPOAEs in 0.1-octave steps of f(2) with primary tones at L(1)=L(2) =55, 65, and 75dB SPL. CBA/CaJ mice exhibited average levels of approximately 26dB SPL and this strain was selected as the normal reference strain against which the others were compared. Based upon the configurations of their DP-grams, the 28 mouse strains could be categorized into four distinct groups. That is, nine of the strains including the CBA were designated as the CBA-like group because these mice displayed robust DPOAE levels across frequency. In contrast, the remaining three groups all exhibited irregular DP-gram patterns. Specifically, eight of the remaining 19 strains showed a progressive high- to low-frequency reduction in DPOAE levels that was typical of age-related hearing loss (AHL) associated with mouse strains homozygous for the ahl allele and were labeled as AHL-like strains. Seven strains demonstrating relatively even patterns of reduced DPOAE levels across the frequency-test range were designated as Flat-loss strains. Finally, the remaining four strains exhibited no measurable DPOAEs at either 3 or 5 mo of age and thus were classified as Absent strains. Extending the f(2) test frequencies up to approximately 54kHz led to the detection of very early-onset reductions in cochlear function in non-CBA-like groups so that all strains could be categorized by 3 mo of age. Predictably, the AHL-like strains showed more pronounced DPOAE losses at 5 mo than at 3 mo. A similar deterioration in DPOAE levels was not apparent for the Flat-loss strains. Both the AHL-like and Flat-loss strains showed considerably more variability in DPOAE levels than did the CBA-like strains. Together, these findings indicate that DP-grams adequately reveal both frequency-specific loss patterns and details of inbred strain variability.

Psychophysical, basilar-membrane (BM), and single nerve-fiber tuning curves, as well as suppression of distortion-product otoacoustic emissions (DPOAEs), all give rise to frequency tuning patterns with stereotypical features. Similarities and differences between the behaviors of these tuning functions, both in normal conditions and following various cochlear insults, have been documented. While neural tuning curves (NTCs) and BM tuning curves behave similarly both before and after cochlear insults known to disrupt frequency selectivity, DPOAE suppression tuning curves (STCs) do not necessarily mirror these responses following either administration of ototoxins [Martin et al., J. Acoust. Soc. Am. 104, 972-983 (1998)] or exposure to temporarily damaging noise [Howard et al., J. Acoust. Soc. Am. 111, 285-296 (2002)]. However, changes in STC parameters may be predictive of other changes in cochlear function such as cochlear immaturity in neonatal humans [Abdala, Hear. Res. 121, 125-138 (1998)]. To determine the effects of noise-induced permanent auditory dysfunction on STC parameters, rabbits were exposed to high-level noise that led to permanent reductions in DPOAE level, and comparisons between pre- and postexposure DPOAE levels and STCs were made. Statistical comparisons of pre- and postexposure STC values at CF revealed consistent basal shifts in the frequency region of greatest cochlear damage, whereas thresholds, Q10dB, and tip-to-tail gain values were not reliably altered. Additionally, a large percentage of high-frequency lobes associated with third tone interference phenomena, that were exhibited in some data sets, were dramatically reduced following noise exposure. Thus, previously described areas of DPOAE interference above f2 may also be studied using this type of experimental manipulation [Martin et al., Hear. Res. 136, 105-123 (1999); Mills, J. Acoust. Soc. Am. 107, 2586-2602 (2002)].

Ephrins and Eph receptors are a family of molecules that have been implicated in many developmental processes including neuronal network formation, guidance of cell migration, and axonal pathfinding. These molecules exhibit the ability to send bidirectional signals following ligand–receptor interactions resulting from cell–cell contacts. Gene-targeted knockout mice of B-class ephrins and Eph receptors have been shown to display phenotypic responses that correlate with anatomical defects. For example, disruption of the EphB2 receptor leads to defects of the vestibular system, including pathfinding abnormalities in efferent axons and reduced endolymph production. Such developmental distortions lead to deficiencies in ionic homeostasis and repetitive circling behaviors. The present study demonstrates that B-class ephrins and Eph receptors are expressed in cochlear tissues, suggesting that they may play some role in auditory function. To determine whether ephrins and Eph receptors have a functional role in the peripheral auditory system, distortion-product otoacoustic emission (DPOAE) levels, collected across a broad frequency range, were compared between groups of mice expressing different Eph receptor genotypes. In particular, EphB1 and EphB3 receptor knockout mice exhibited significantly diminished DPOAE levels as compared to wild-type littermates, indicating that these specific Eph receptors are necessary for normal cochlear function.

Although the distinction between "biased" and "unbiased" is generally recognized as an important methodological issue in place conditioning, previous studies have not adequately addressed the distinction between a biased/unbiased apparatus and a biased/unbiased stimulus assignment procedure. Moreover, a review of the recent literature indicates that many reports (70% of 76 papers published in 2001) fail to provide adequate information about apparatus bias. This issue is important because the mechanisms underlying a drug's effect in the place-conditioning procedure may differ depending on whether the apparatus is biased or unbiased. The present studies were designed to assess the impact of apparatus bias and stimulus assignment procedure on ethanol-induced place conditioning in mice (DBA/2 J). A secondary goal was to compare various dependent variables commonly used to index conditioned place preference. Apparatus bias was manipulated by varying the combination of tactile (floor) cues available during preference tests. Experiment 1 used an unbiased apparatus in which the stimulus alternatives were equally preferred during a pre-test as indicated by the group average. Experiment 2 used a biased apparatus in which one of the stimuli was strongly preferred by most mice (mean % time on cue = 67%) during the pre-test. In both studies, the stimulus paired with drug (CS+) was assigned randomly (i.e., an "unbiased" stimulus assignment procedure). Experimental mice received four pairings of CS+ with ethanol (2 g/kg, i.p.) and four pairings of the alternative stimulus (CS−) with saline; control mice received saline on both types of trial. Each experiment concluded with a 60-min choice test. With the unbiased apparatus (experiment 1), significant place conditioning was obtained regardless of whether drug was paired with the subject's initially preferred or non-preferred stimulus. However, with the biased apparatus (experiment 2), place conditioning was apparent only when ethanol was paired with the initially non-preferred cue, and not when it was paired with the initially preferred cue. These conclusions held regardless of which dependent variable was used to index place conditioning, but only if the counterbalancing factor was included in statistical analyses. These studies indicate that apparatus bias plays a major role in determining whether biased assignment of an ethanol-paired stimulus affects ability to demonstrate conditioned place preference. Ethanol's ability to produce conditioned place preference in an unbiased apparatus, regardless of the direction of the initial cue bias, supports previous studies that interpret such findings as evidence of a primary rewarding drug effect. Moreover, these studies suggest that the asymmetrical outcome observed in the biased apparatus is most likely due to a measurement problem (e.g., ceiling effect) rather than to an interaction between the drug's effect and an unconditioned motivational response (e.g., "anxiety") to the initially non-preferred stimulus. More generally, these findings illustrate the importance of providing clear information on apparatus bias in all place-conditioning studies.

Distortion-product otoacoustic emissions (DPOAEs) at 2f1-f2 can be suppressed by the introduction of a third "suppressor" tone. Plotting the suppression of the DPOAE level against the changing frequency and level of the suppressor produces frequency-tuning functions referred to as suppression tuning curves (STCs). The dominant features of STCs, including their shape, are similar to the features of neural tuning curves (NTCs) recorded from single auditory nerve fibers. However, recent findings using reversible diuretics suggest that STCs do not provide the same measure of cochlear frequency selectivity as provided by NTCs. To determine if STCs are also insensitive to the adverse effects of excessive sounds, the present study exposed rabbits to a moderate-level noise that produced temporary threshold shift-like (TTS) effects on DPOAEs, and examined the influence of such exposures on STCs. DPOAEs were produced using primary tones with geometric-mean frequencies centered at 2.8 or 4 kHz, and with L1 and L2 values of 45/45, 50/35, 50/50, and 55/45 dB SPL. STCs were obtained before and during recovery for a period of approximately 2 h immediately following, and at 1, 2, 3, and 7 d post-exposure to a 2 kHz octave band noise, at levels and durations sufficient to cause significant but reversible reductions in DPOAE levels. STC data included tip center frequency, tip threshold, and Q10dB measures of tuning for suppression criteria of 3, 6, 9, and 12 dB. Recovery was variable between animals, but all rabbits recovered fully by 7 d post-exposure. STC center frequencies measured during the TTS typically tuned to a slightly higher frequency, while tip thresholds tended to decrease and Q10dB increase. Together, the results indicate that, despite similarities in the general properties of STCs and NTCs, these two types of tuning curves are affected differently following reversible cochlear insult.