Intraoperative fibrin formation during Descemet membrane endothelial keratoplasty.

PURPOSE: To describe Descemet membrane endothelial keratoplasty (DMEK) cases complicated by spontaneous intraoperative fibrin formation. METHODS: DMEK surgeries performed at two centers using a standardized technique were reviewed retrospectively for the occurrence of intraoperative fibrin formation. Cases were assessed for recipient medical history, donor age, best spectacle-corrected visual acuity (BSCVA), intraoperative unscrolling time, 6-month endothelial cell loss (ECL), and the course of the mate donor cornea. RESULTS: In this review of 868 cases of standardized DMEK surgery with surgical peripheral iridotomy, 32 eyes of 29 patients (3.7%) were complicated by the formation of intraoperative fibrin formation, including 3 patients that developed fibrin in both eyes. Three of the 32 grafts failed (9.4%). None of the mate corneas transplanted (n = 27) developed complications related to fibrin. The donor age ranged from 51 to 75 years and recipient age ranged from 49 to 82 years (median, 66 years). Unscrolling time ranged from 1 to 105 min (median, 15 min). Nine eyes required one rebubble procedure. No eyes had vision-limiting comorbidities, and the 6-month BSCVA was ≥20/40 in all eyes. Six-month ECL ranged from 19% to 73% (median, 44%). CONCLUSIONS: We conclude that fibrin formation during DMEK surgery is an uncommon but important complication that can make graft manipulation more difficult, and may have deleterious effects on endothelial cell density and graft survival.

Reports of Post-keratoplasty Infections for Eye Bank-prepared and Non-Eye Bank-prepared Corneas: 12 Years of Data From a Single Eye Bank.

PURPOSE: To examine postoperative reports of infection rates for eye bank-prepared and non-eye bank-prepared corneas from January 1, 2006 to December 31, 2017, from a single eye bank. METHOD: A retrospective review of reported fungal and bacterial infections with corneal transplant surgeries using corneas distributed by our eye bank was conducted. The reported number of infections for corneas that underwent eye bank preparation (pre-cut and pre-stripped corneas) and for those distributed without eye bank preparation was quantified. The potential association between infection rates in tissue prepared by the eye bank and those in corneas that had no additional eye bank processing was also examined. RESULTS: Four of 17,035 corneas distributed during the study period were associated with fungal infections (1 eye bank-prepared and 3 non-eye bank-prepared corneas) and were attributed to the tissue after investigation by eye bank medical directors. There was no ascending trend of infections reported with eye bank-prepared corneas in the first 3 years (2 of 1054 corneas, 0.19%) compared with that in the last 3 years of the study period (6 of 3500 corneas, 0.17%; P = 0.901) when the eye bank distributed 3 times more prepared corneas than non-eye bank-prepared corneas. A significant increase in the numbers of reported infections for non-eye bank-prepared corneas was observed between these 3-year intervals (0.1% in the first 3 years to 1.58% in the last 3 years; P = 0.001). CONCLUSIONS: Reports of infections remained low despite increased use of eye bank-prepared tissue. These results suggest that factors other than eye bank tissue preparation should be considered when investigating potential sources of pathogen contamination in donor corneas.

Preloaded Descemet Membrane Endothelial Keratoplasty Donor Tissue: Surgical Technique and Early Clinical Results.

PURPOSE: To describe the technique, advantages, and early complication rates of using Descemet membrane endothelial keratoplasty (DMEK) donor tissue that is prestained and preloaded into an injector at the eye bank and delivered in a storage medium to the surgeon for transplantation 1 to 2 days later. METHODS: A total of 111 eyes with endothelial failure underwent DMEK using donors that were prestripped, prestained, S-stamped, and preloaded into a Straiko modified Jones tube and delivered in an Optisol-filled viewing chamber 1 to 2 days later. Scroll tightness, time to unscroll and center the tissue, postoperative rebubble rate, and graft failure rate were recorded. Endothelial cell density was measured at 3 and 6 months. RESULTS: All tissues remained well stained with easy visualization at the time of surgery (n = 111). The mean scroll tightness was 2.2 (range: 1-4). The mean time to center and unscroll the tissue was 3.5 minutes (range: 0.5-11.25 min). There was no primary graft failure. There were 16 cases with the placement of another bubble postoperatively (with a 14.4% rebubble rate). Of those 16 cases, 2 required a second rebubble. Endothelial cell loss at 3 and 6 months postoperatively was 26.7% (n = 63 eyes) and 30.9% (n = 67 eyes), respectively. CONCLUSIONS: This is the first report of the clinical use of prestained, preloaded tissue for DMEK. The characteristics and handling of the tissue were not different from those of surgeon-loaded tissue. Because punching, staining, and loading the graft intraoperatively is not necessary, the surgery time and risk of damaging donor tissue are reduced when using preloaded tissue.

Descemet Membrane Endothelial Keratoplasty in Eyes With Previous Laser Refractive Surgery: Outcomes and Complications.

PURPOSE: To evaluate the refractive predictability and efficacy of Descemet membrane endothelial keratoplasty (DMEK) for patients with previous laser refractive surgery. METHODS: We retrospectively reviewed our cohort of endothelial keratoplasty surgical cases. We identified 21 eyes that underwent laser-assisted in situ keratomileusis (n = 17) or photorefractive keratectomy (n = 4) and were treated with DMEK for endothelial failure. Patients were analyzed preoperatively and at 6 months postoperatively for changes in visual acuity, refraction, and corneal topography. RESULTS: Six months after surgery, eyes significantly improved to a mean best-corrected visual acuity of 20/23 (P < 0.001). Mean astigmatism amplitude changed from 1.13 ± 0.96 preoperatively to 0.92 ± 0.51 diopters postoperatively (P = 0.28). However, shifts in the axis of corneal astigmatism ranged from 1 to 70 degrees, with 6 eyes (30%) showing an axis shift of more than 30 degrees. Spherical equivalents in nontriple procedures remained unchanged (n = 16; P = 0.69) at 6 months. CONCLUSIONS: DMEK and DMEK triple procedures are predictable in patients with previous refractive surgery achieving good visual results. However, refraction after the use of toric intraocular lenses may be unpredictable because of the variability in changes of the magnitude and axis of corneal astigmatism; we recommend extreme caution in the use of the toric intraocular lens in this group of patients and proper counseling for possible individual postoperative residual astigmatism.

Intraoperative S-Stamp Enabled Rescue of 3 Inverted Descemet Membrane Endothelial Keratoplasty Grafts.

PURPOSE: To report the clinical outcomes of 3 Descemet membrane endothelial keratoplasty (DMEK) grafts, in which an upside down DMEK graft orientation was discovered after SF6 gas insufflation of the anterior chamber and the orientation was then corrected intraoperatively. METHODS: A total of 371 consecutive DMEK cases using an S-stamp were analyzed and found to include 3 cases in which the DMEK graft was initially elevated with SF6 gas in an upside down orientation, as demonstrated intraoperatively by the S-stamp. In each case, the graft was immediately manipulated into the proper orientation and the rescued case completed using our standard technique. The postoperative complications of these cases were reported and the percentage of endothelial cell loss at 6 months was compared with that of cases in this series without this rare intraoperative complication. RESULTS: All 3 rescued grafts achieved successful recipient corneal clearance and improved vision for the patient. There was no graft failure, rebubble, or rejection in these 3 cases. The average 6-month endothelial cell loss in the 3 initially inverted grafts was 51%, 58.3%, and 67% compared with 31.6% (SD ± 17.4%) in the series at large. CONCLUSIONS: Incorporation of a stromal-sided S-stamp has been previously shown to reduce the risk of iatrogenic primary graft failure through the prevention of upside down graft implantation. In these 3 instances, although the stamp did not prevent initial upside down graft elevation, it did allow the surgeon to immediately recognize this rare problem, correct it intraoperatively, and prevent iatrogenic graft failure in each case.

Astigmatism Correction With Toric Intraocular Lenses in Descemet Membrane Endothelial Keratoplasty Triple Procedures.

PURPOSE: To report the clinical efficacy of astigmatism correction with toric intraocular lenses (IOLs) in patients undergoing the Descemet membrane endothelial keratoplasty (DMEK) triple procedure and to evaluate the accuracy of the correction. METHODS: Fifteen eyes of 10 patients who received cataract extraction, toric IOL placement, and DMEK surgery for Fuchs corneal dystrophy and cataracts were evaluated. The cylinder power of toric IOLs was determined by an online toric calculator with keratoscopy measurements obtained using Scheimpflug corneal imaging. Prediction errors were assessed as a difference vector between the anticipated minus postoperative residual astigmatism. RESULTS: At 10.1 ± 4.9 months postoperatively, 8/13 (61.5%) of eyes achieved uncorrected distance visual acuity better than 20/40. Mean best spectacle-corrected distance visual acuity (logMAR) improved from 0.21 ± 0.15 preoperatively to 0.08 ± 0.12 postoperatively (P < 0.01). The magnitude of refractive astigmatism was also significantly decreased from 2.23 ± 1.10 D (range 0.75-4.25 D) preoperatively to 0.87 ± 0.75 D (range 0.00-3.00 D) postoperatively (P < 0.01). In 1 eye with rotational misalignment by 43 degrees, we found no improvement of astigmatism. The prediction error of astigmatism at the corneal plane was 0.77 ± 0.54 D (range 0.10-1.77 D). Four eyes with preoperative "with-the-rule" corneal astigmatism had postoperative "against-the-rule" refractive astigmatism. CONCLUSIONS: For patients with Fuchs corneal dystrophy and cataracts, use of toric IOLs might be a valuable option in triple DMEK surgery. Additionally, care should be taken to prevent excessive IOL rotation.

Donor Endothelial Cell Density Measurements Do Not Change Immediately After DMEK Preparation.

PURPOSE: To evaluate a single eye bank's measurement of endothelial cell density (ECD) of Descemet membrane endothelial keratoplasty (DMEK) grafts before and after preparation using 2 separate counting methods. METHODS: A series of 60 donor tissues were prepared for DMEK surgery. One to 4 specular images of the central endothelium were taken both before and after preparation, and ECDs were evaluated for a total of 345 unique images. Images were then masked and provided to the Cornea Image Analysis Reading Center (CIARC) for independent analysis. RESULTS: Before preparation, average eye bank-determined ECD with the center method was 2678 ± 259 cells/mm and was 2599 ± 280 cells/mm CIARC-determined by the variable frame method (P < 0.001, n = 176). After preparation, eye bank-determined ECD was 2719 ± 265 cells/mm and CIARC-determined ECD was 2615 ± 344 cells/mm (P < 0.001, n = 169). The difference in ECD before and after DMEK preparation was not found to be statistically significant when evaluated using either analysis method (P = 0.19; P = 0.64) before and after preparation, respectively. CONCLUSIONS: Although the absolute ECD value may differ by the analysis method statistically, pre- and post-DMEK preparation ECDs did not significantly change by either analysis method. Other methods such as vital staining to assess tissue damage after preparation in conjunction with specular microscopy are suggested.

Corneal Astigmatism Stability in Descemet Membrane Endothelial Keratoplasty for Fuchs Corneal Dystrophy.

PURPOSE: To calculate the magnitude and angle of the shift in corneal astigmatism associated with Descemet membrane endothelial keratoplasty (DMEK) surgery to determine the feasibility of concurrent astigmatism correction at the time of DMEK triple procedures. DESIGN: Retrospective study. METHODS: Forty-seven eyes that previously underwent the DMEK procedure for Fuchs endothelial corneal dystrophy and that had more than 1.0 diopter (D) of front corneal astigmatism preoperatively were identified. All DMEK surgeries used a clear corneal temporal incision of 3.2 mm. Surgically induced astigmatism (SIA) was evaluated 6 months postsurgery with vector analysis using Scheimpflug image reading. RESULTS: We did not find a difference between pre- and postoperative magnitude of front astigmatism (P = 0.88; paired t test). The magnitude of the SIA front surface was 0.77 ± 0.63 D (range, 0.10-3.14 D). The centroid vector of the SIA front surface was 0.14 at 89.3°. A hyperopic corneal power shift was noted in both the front surface by 0.26 ± 0.74 D (range, 0.45-3.05 D) (P = 0.018; paired t test) and back surface by 0.56 ± 0.55 D (range, 0.25-2.40 D) (P < 0.01; paired t test). CONCLUSIONS: DMEK surgery induces minimal amounts of corneal astigmatism that is a with-the-rule shift associated with a temporal clear corneal incision. The stability of these data from preop to postop supports the plausibility of incorporating astigmatism correction with the cautious use of toric intraocular lenses for patients with Fuchs corneal dystrophy and cataract.

Descemet Membrane Endothelial Keratoplasty (DMEK) Tissue Preparation: A Donor Diabetes Mellitus Categorical Risk Stratification Scale for Assessing Tissue Suitability and Reducing Tissue Loss.

PURPOSE: This study assessed a novel diabetes mellitus (DM) rating scale in relation to its utility in reducing Descemet membrane endothelial keratoplasty (DMEK) tissue preparation failure. METHODS: A 5-point DM rating scale was defined, in which 1 demonstrated relatively good health associated with DM and 5 represented comorbidities associated with DM. A chart review from consecutive donors who had at least 1 tissue prepared for DMEK was performed. Using the donor profile, the first tissue processed from each donor was categorized according to the DM severity and if the tissue passed or failed the DMEK preparation. Failure rates per rating group were evaluated using logistic regression and odds of preparation failure. RESULTS: A total of 125 tissues prepared for DMEK were categorized based on the defined DM rating scale. Of these, 9 tissues were rated 1 (11.1% failure), 25 were rated 2 (0% failure), 31 were rated 3 (6.5% failure), 24 were rated 4 (16.7% failure), and 36 were rated 5 (30.6% failure). The odds ratios were significant for tissues rated as 5 and 3 (P < 0.05). No other rating categories were found to influence the odds of failure. A χ test comparing categories of low risk (1-3) and high risk (4-5) was also performed (P = 0.001). CONCLUSIONS: The DM rating scale does seem to stratify the risk of preparation failure associated with the severity of DM and associated comorbidities. Inclusion of some diabetic donors for the preparation of DMEK grafts may be warranted given proper screening of the donor history and application of the rating scale.

Relationship Between Tissue Unscrolling Time and Endothelial Cell Loss.

PURPOSE: To assess the relationship between intraoperative unscrolling time of the donor Descemet membrane endothelial keratoplasty (DMEK) tissue and 6-month postoperative endothelial cell loss (ECL), and to determine whether donor age, scroll tightness, and the presence of an S stamp are related to unscrolling time. METHODS: Ninety-three consecutive uncomplicated DMEK surgeries performed on eyes with Fuchs endothelial dystrophy using our standardized technique (ie, prestripped tissue with or without a premarked S stamp from our eye bank, overstripping the recipient, Straiko glass injector, no-touch tap technique, and bubble of 20% SF6 gas) were evaluated. Intraoperative unscrolling times and 6-month endothelial cell densities were measured and analyzed. RESULTS: Sixty-nine cases comprised the study cohort. The median unscrolling time was 4 minutes (range: 0.8-17.5 minutes), and the median ECL was 26.9% (range: -4.3% to 80.0%). There was no relationship between unscrolling time and ECL at 6 months by the Pearson correlation coefficient (r = -0.02, P = 0.89). Younger donor age, tighter scrolls, and absence of an S stamp had no correlation with longer unscrolling times (all P > 0.05). Only 2 of 4 cases of iatrogenic primary graft failure had unscrolling times available for analysis; in this limited sample, there was no association between iatrogenic primary graft failure and unscrolling time. CONCLUSIONS: Once the DMEK tissue is safely in the anterior chamber, surgeons need not rush the "DMEK dance" because longer unscrolling times may not endanger the endothelium.

The S-stamp in Descemet Membrane Endothelial Keratoplasty Safely Eliminates Upside-down Graft Implantation.

PURPOSE: To present 6-month clinical outcomes from a series of 165 consecutive Descemet membrane endothelial keratoplasty (DMEK) procedures before and after the introduction of a novel stromal-sided S-stamp preparation technique that has decreased the incidence of iatrogenic primary graft failure by eliminating upside-down grafts. DESIGN: Retrospective nonrandomized comparative case series. PARTICIPANTS: We included 165 consecutive eyes that had undergone DMEK surgery for Fuchs' or pseudophakic bullous keratopathy. These cases were divided into 2 cohorts: the first cohort comprised 31 cases that used unstamped tissue before the S-stamp was introduced, and the second cohort comprised 133 cases after the S-stamp was incorporated into the standardized technique. A single unstamped DMEK case was performed after the introduction of the S-stamp for a total of 32 unstamped cases. METHODS: Donor materials were prepared at a single eye bank using a standardized technique, which subsequently incorporated the addition of a dry ink gentian violet S-stamp to the stromal side of Descemet membrane. All surgeries were performed at a single clinical site by 5 surgeons (2 attending surgeons and 3 fellows). Two of the 165 DMEK cases were performed for pseudophakic bullous keratopathy (2 cases, 1 in each cohort), and the remaining cases were for Fuchs' endothelial dystrophy. Primary outcome measures were assessed at 6 months and maintained in a prospective institutional review board-approved study. MAIN OUTCOME MEASURES: We analyzed the 6-month endothelial cell density, incidence of iatrogenic primary graft failure, upside-down graft implantation, and rebubble events. RESULTS: The S-stamp eliminated upside-down graft implantations (0/133 S-stamped vs 3/32 unstamped) and did not significantly alter 6-month endothelial cell loss (31±17% S-stamped vs 29±14% unstamped; P = 0.62) or frequency of rebubble (17/133 S-stamped vs 1/32 unstamped; P = 0.20). CONCLUSION: The incorporation of a stromal-sided S-stamp eliminates iatrogenic primary graft failure owing to upside-down implantation of DMEK grafts, without adversely affecting early postoperative complications or 6-month endothelial cell loss.

The First 100 Eyes of Standardized Descemet Stripping Automated Endothelial Keratoplasty versus Standardized Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To compare results of the first 100 eyes of Descemet stripping automated endothelial keratoplasty (DSAEK) and Descemet membrane endothelial keratoplasty (DMEK) that were performed with a standardized technique at a single institution. DESIGN: Single-center, retrospective case series. PARTICIPANTS: The first 100 eyes of standardized DSAEK and DMEK that underwent surgery for Fuchs corneal dystrophy at our center. We excluded patients with prior ocular surgery other than cataract surgery to limit confounding variables. METHODS: Best spectacle-corrected visual acuity (BSCVA; in logarithm of the minimal angle of resolution [logMAR] units) was obtained and specular microscopy of donor corneal tissue was performed before surgery. Postoperative complications, BSCVA, and the percent of endothelial cell loss (ECL) recorded at 6 months were compared with the Student t test. Patients with pre-existing ocular comorbidity that impacted visual potential such as macular degeneration, amblyopia, advanced glaucoma, and other optic neuropathies were excluded from the analysis of visual acuity, but were included for the outcomes of complications and ECL. MAIN OUTCOME MEASURES: Visual acuity improvement, ECL 6 months after surgery, postoperative complications, iatrogenic primary graft failure (IPGF), and rebubbling. RESULTS: Of the 200 eyes, 62 DSAEK eyes and 70 DMEK eyes had 6-month BSCVA available and no vision-limiting comorbidities. Mean BSCVA increased from 0.41±0.19 logMAR and 0.27±0.11 logMAR before surgery to 0.20±0.13 logMAR and 0.11±0.13 logMAR 6 months after DSAEK and DMEK, respectively (P<0.001). Seventy-one DSAEK eyes and 70 DMEK eyes had 6-month ECL data available: ECL was 25.9±14.0% after DSAEK and 27.9±16.0% after DMEK (P=0.38). There were no IPGFs in the DSAEK cohort and there were 4 of 100 IPGFs after DMEK (P=0.12). Rebubbling was performed in 2 of 100 eyes after DSAEK and in 6 of 100 eyes after DMEK (P=0.28). CONCLUSIONS: Compared with DSAEK, DMEK provided better visual recovery and comparable 6-month ECL. The DMEK group had a higher, although not statistically significant, percentage of rebubbling procedures and IPGFs.

Stamping an S on DMEK Donor Tissue to Prevent Upside-Down Grafts: Laboratory Validation and Detailed Preparation Technique Description.

PURPOSE: To report endothelial cell loss (ECL) caused by a novel S-stamp preparation technique for Descemet membrane endothelial keratoplasty (DMEK). METHODS: Six cadaveric human corneas were prepared for DMEK transplantation using a single standardized technique, including the application of a dry ink gentian violet S-stamp to the stromal side of Descemet membrane. Endothelial cell death was evaluated and quantified using computerized analysis of vital dye staining. RESULTS: ECL caused by the S-stamp was 0.6% (range 0.1%-1.0%), which comprised less than one-tenth of the total ECL caused by our preparation of the DMEK graft from the start to finish, including recovery, prestripping, S-stamping, and trephination (13.7% total ECL, range 9.9%-17.6%). CONCLUSIONS: Our novel S-stamp donor tissue preparation technique is intuitive to learn and holds the promise of preventing iatrogenic primary graft failure due to upside-down grafts without causing unacceptable increases in ECL.

Standardized DMEK Technique: Reducing Complications Using Prestripped Tissue, Novel Glass Injector, and Sulfur Hexafluoride (SF6) Gas.

PURPOSE: To report low complication rates in Descemet membrane endothelial keratoplasty (DMEK) using sulfur hexafluoride (SF6) gas, a novel glass injector, and donor tissue prestripped by an eye bank technician. METHODS: A standardized technique of DMEK was performed in 80 consecutive Fuchs corneal dystrophy cases using technician-prestripped tissue, a novel glass injector, a modified Yoeruek tap technique, and an SF6 gas (20% concentration) bubble for prolonged tissue support. Twenty-five donors were premarked with an "S" stamp for intraoperative orientation. Surgery was performed by 2 experienced DMEK surgeons and 2 inexperienced cornea fellows. Complications were recorded, and the percent endothelial cell loss was calculated at 6 months postoperatively. RESULTS: There were 5 cases that received an air bubble injection postoperatively (6% rebubble rate). There were 6 grafts that immediately failed, 2 because of excessive surgical trauma, and 4 because of upside-down graft placement documented by optical coherence tomography. None of the 25 cases with an S stamp failed. Recipient corneas cleared quickly with no clinical evidence of toxicity from the SF6 gas bubble, and the grafts experienced a mean endothelial cell loss of 27% at 6 months. CONCLUSIONS: Tissue prestripped by an eye bank technician can be safely used for DMEK surgery. SF6 gas for prolonged tissue support may reduce the rebubble rate in DMEK, with no apparent acute toxic effect. An unrecognized upside-down graft was the primary cause of graft failure in this series. Upside-down grafts may be eliminated by the use of donor tissue premarked by the eye bank with an S orientation stamp.

Inhibition does not affect the timing code for vocalizations in the mouse auditory midbrain.

Many animals use a diverse repertoire of complex acoustic signals to convey different types of information to other animals. The information in each vocalization therefore must be coded by neurons in the auditory system. One way in which the auditory system may discriminate among different vocalizations is by having highly selective neurons, where only one or two different vocalizations evoke a strong response from a single neuron. Another strategy is to have specific spike timing patterns for particular vocalizations such that each neural response can be matched to a specific vocalization. Both of these strategies seem to occur in the auditory midbrain of mice. The neural mechanisms underlying rate and time coding are unclear, however, it is likely that inhibition plays a role. Here, we examined whether inhibition is involved in shaping neural selectivity to vocalizations via rate and/or time coding in the mouse inferior colliculus (IC). We examined extracellular single unit responses to vocalizations before and after iontophoretically blocking GABAA and glycine receptors in the IC of awake mice. We then applied a number of neurometrics to examine the rate and timing information of individual neurons. We initially evaluated the neuronal responses using inspection of the raster plots, spike-counting measures of response rate and stimulus preference, and a measure of maximum available stimulus-response mutual information. Subsequently, we used two different event sequence distance measures, one based on vector space embedding, and one derived from the Victor/Purpura D q metric, to direct hierarchical clustering of responses. In general, we found that the most salient feature of pharmacologically blocking inhibitory receptors in the IC was the lack of major effects on the functional properties of IC neurons. Blocking inhibition did increase response rate to vocalizations, as expected. However, it did not significantly affect spike timing, or stimulus selectivity of the studied neurons. We observed two main effects when inhibition was locally blocked: (1) Highly selective neurons maintained their selectivity and the information about the stimuli did not change, but response rate increased slightly. (2) Neurons that responded to multiple vocalizations in the control condition, also responded to the same stimuli in the test condition, with similar timing and pattern, but with a greater number of spikes. For some neurons the information rate generally increased, but the information per spike decreased. In many of these neurons, vocalizations that generated no responses in the control condition generated some response in the test condition. Overall, we found that inhibition in the IC does not play a substantial role in creating the distinguishable and reliable neuronal temporal spike patterns in response to different vocalizations.

Inhibition shapes selectivity to vocalizations in the inferior colliculus of awake mice.

The inferior colliculus (IC) is a major center for integration of auditory information as it receives ascending projections from a variety of brainstem nuclei as well as descending projections from the thalamus and auditory cortex. The ascending projections are both excitatory and inhibitory and their convergence at the IC results in a microcircuitry that is important for shaping responses to simple, binaural, and modulated sounds in the IC. Here, we examined the role inhibition plays in shaping selectivity to vocalizations in the IC of awake, normal-hearing adult mice (CBA/CaJ strain). Neurons in the IC of mice show selectivity in their responses to vocalizations, and we hypothesized that this selectivity is created by inhibitory microcircuitry in the IC. We compared single unit responses in the IC to pure tones and a variety of ultrasonic mouse vocalizations before and after iontophoretic application of GABA(A) receptor (GABA(A)R) and glycine receptor (GlyR) antagonists. The most pronounced effects of blocking GABA(A)R and GlyR on IC neurons were to increase spike rates and broaden excitatory frequency tuning curves in response to pure tone stimuli, and to decrease selectivity to vocalizations. Thus, inhibition plays an important role in creating selectivity to vocalizations in the IC.

Preparation of an awake mouse for recording neural responses and injecting tracers.

It is well known that anesthesia alters neural response properties in various regions of the brain. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats to mouse. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.