Early outcomes after definitive chemoradiation therapy with Vienna/Venezia hybrid high-dose rate brachytherapy applicators for cervical cancer: A single-institution experience.

PURPOSE: The Vienna and Venezia (Elekta) are hybrid intracavitary/interstitial brachytherapy (BT) applicators for cervical cancers unsuitable for intracavitary BT alone to improve target coverage or reduce critical organ dose. There is limited outcome data with the use of these applicators outside published experience of the EMBRACE group. We report feasibility and early outcomes with the use of these hybrid applicators at our institution. METHODS AND MATERIALS: Hybrid applicators were used to treat 61 patients with cervical cancer from November 2011 to December 2019. Indications for hybrid applicator use were involvement of the vagina in 10 patients (16%), residual central or parametrial disease in 46 patients (75%), and a narrow introitus in 5 patients (9%). Toxicities were graded using the CTCAE v4.0. Outcomes were assessed with the Kaplan-Meier method. RESULTS: Median follow-up was 16 months (IQR 9-32 mos). Median HRCTV volume was 31.6 cm3 (IQR 25-48 cm3). Median HRCTV D90 was 86.1 Gy (IQR 84.3-88.0 Gy). In 54 patients with follow-up PET/CT at 3 months, complete initial imaging response locally was seen in 46 patients.Estimated 12-month Kaplan-Meier overall survival, locoregional control, distant control, and recurrence-free survival estimates were 86.9%, 80.6%, 73.8%, and 65.9%, respectively. The 12-month incidence of Grade 3+ GI/GU chronic toxicities was 5.7%, consisting of vesicovaginal fistula, rectovaginal fistula, and ureterovesical fistula. CONCLUSIONS: Our single-institution data support the use of the hybrid applicators, as an alternative to traditional BT applicators when clinically warranted. Use of hybrid applicators is feasible with adequate coverage of disease in the vagina and parametrium.

Evaluation of set-up uncertainties with daily kilovoltage image guidance in external beam radiation therapy for gynaecological cancers.

AIMS: To assess the set-up uncertainty for gynaecological cancer patients treated with external beam radiation therapy using daily kilovoltage image guidance and to estimate set-up margins for treatment and factors that would predict higher set-up uncertainty. MATERIALS AND METHODS: Alignment data from daily two-dimensional kilovoltage planar images and three-dimensional kilovoltage cone beam images for 52 patients treated on a Varian 2300iX linear accelerator with On Board Imaging (OBI; version 1.4) capability were analysed. The mean displacements of translational shifts, population systematic errors and random errors were calculated. Using van Herk's formula, the clinical target volume (CTV) to planning target volume (PTV) margins for set-up uncertainties were calculated. The differences in set-up error were calculated with respect to the type of cancer, imaging type and body mass index (BMI). RESULTS: Population systematic and random errors were 1.1 mm, 2.3 mm, 2.3 mm and 3.9 mm, 5.0 mm, 3.5 mm in the anterior-posterior (AP), medial-lateral (ML) and superior-inferior (SI) directions, respectively, for the entire patient population. Using van Herk's formula, the CTV to PTV margins for set-up uncertainties were found to be 5.5, 9.1 and 8.3 mm in the AP, ML and SI directions respectively. The mean displacements in the AP, ML and SI directions for BMI ≥ 30 (28 patients) versus <30 (24 patients) were -0.1 mm, 0.9 mm and 1.0 mm versus -0.1 mm, 0.1 mm and 0.4 mm, respectively, (P = 0.02). CONCLUSIONS: Daily imaging helps to assess set-up uncertainty. The set-up margin for CTV to PTV was larger for patients with BMI ≥ 30 without image guidance and these patients would benefit more from daily image guidance.

Increased cholecystokinin labeling in the hippocampus of a mouse model of epilepsy maps to spines and glutamatergic terminals.

The neuropeptide cholecystokinin (CCK) is abundant in the CNS and is expressed in a subset of inhibitory interneurons, particularly in their axon terminals. The expression profile of CCK undergoes numerous changes in several models of temporal lobe epilepsy. Previous studies in the pilocarpine model of epilepsy have shown that CCK immunohistochemical labeling is substantially reduced in several regions of the hippocampal formation, consistent with decreased CCK expression as well as selective neuronal degeneration. However, in a mouse pilocarpine model of recurrent seizures, increases in CCK-labeling also occur and are especially striking in the hippocampal dendritic layers of strata oriens and radiatum. Characterizing these changes and determining the cellular basis of the increased labeling were the major goals of the current study. One possibility was that the enhanced CCK labeling could be associated with an increase in GABAergic terminals within these regions. However, in contrast to the marked increase in CCK-labeled structures, labeling of GABAergic axon terminals was decreased in the dendritic layers. Likewise, cannabinoid receptor 1-labeled axon terminals, many of which are CCK-containing GABAergic terminals, were also decreased. These findings suggested that the enhanced CCK labeling was not due to an increase in GABAergic axon terminals. The subcellular localization of CCK immunoreactivity was then examined using electron microscopy, and the identities of the structures that formed synaptic contacts were determined. In pilocarpine-treated mice, CCK was observed in dendritic spines and these were proportionally increased relative to controls, whereas the proportion of CCK-labeled terminals forming symmetric synapses was decreased. In addition, CCK-positive axon terminals forming asymmetric synapses were readily observed in these mice. Double labeling with vesicular glutamate transporter 1 and CCK revealed colocalization in numerous terminals forming asymmetric synapses, confirming the glutamatergic identity of these terminals. These data raise the possibility that expression of CCK is increased in hippocampal pyramidal cells in mice with recurrent, spontaneous seizures.

Three-dimensional high dose rate intracavitary image-guided brachytherapy for the treatment of cervical cancer using a hybrid magnetic resonance imaging/computed tomography approach: feasibility and early results.

AIMS: To evaluate the feasibility and outcome of image-guided brachytherapy (IGBT) for treating cervical cancer using magnetic resonance imaging (MRI)-based planning for the first fraction followed by computed tomography (CT)-based planning for subsequent fractions. MATERIALS AND METHODS: Forty-four patients with cervical cancer were treated with three-dimensional high dose rate IGBT. The brachytherapy dose was 5.0-6.0 Gy × five fractions. All but five patients received concurrent weekly cisplatinum at 40 mg/m(2). All patients received external beam radiotherapy (EBRT) with a median dose of 45Gy over 25 fractions. Total doses for the high-risk clinical target volume (HRCTV) and organs at risk, including the rectum, bladder and sigmoid, from EBRT and brachytherapy were summated and normalised to a biologically equivalent dose of 2Gy per fraction (EQD(2)). At 3 months after therapy, any early response was assessed with positron emission tomography (PET)/CT imaging. RESULTS: The mean D(90) for the HRCTV was 83.3 (3.0) Gy. The mean 2 cm (3) dose to the bladder, rectum and sigmoid colon organs was 79.7 (5.1), 57.5 (4.4) and 66.8 (5.7) Gy, respectively. All but one (2.3%) patient had a complete response. Follow-up PET/CT was carried out in 41 (93.0%) patients, of whom 38 (92.5%) had a complete response. Of the 38 patients with a complete response on PET/CT, two had local recurrences at 6 and 8 months, respectively. Actuarial 2 year local control, disease-specific and overall survival rates were 88, 85 and 86%, respectively. CONCLUSION: This is the first report of three-dimensional high dose rate IGBT for the treatment of cervical cancer using a hybrid MRI/CT approach. Early results have shown the feasibility of this approach with excellent local control. Additional studies are needed to assess long-term outcomes of local control and associated morbidities.

Dynamic seizure-related changes in extracellular signal-regulated kinase activation in a mouse model of temporal lobe epilepsy.

Extracellular signal-regulated kinase (ERK) is highly sensitive to regulation by neuronal activity and is critically involved in several forms of synaptic plasticity. These features suggested that alterations in ERK signaling might occur in epilepsy. Previous studies have described increased ERK phosphorylation immediately after the induction of severe seizures, but patterns of ERK activation in epileptic animals during the chronic period have not been determined. Thus, the localization and abundance of phosphorylated extracellular signal-regulated kinase (pERK) were examined in a pilocarpine model of recurrent seizures in C57BL/6 mice during the seizure-free period and at short intervals after spontaneous seizures. Immunolabeling of pERK in control animals revealed an abundance of distinctly-labeled neurons within the hippocampal formation. However, in pilocarpine-treated mice during the seizure-free period, the numbers of pERK-labeled neurons were substantially decreased throughout much of the hippocampal formation. Double labeling with a general neuronal marker suggested that the decrease in pERK-labeled neurons was not due primarily to cell loss. The decreased ERK phosphorylation in seizure-prone animals was interpreted as a compensatory response to increased neuronal excitability within the network. Nevertheless, striking increases in pERK labeling occurred at the time of spontaneous seizures and were evident in large populations of neurons at very short intervals (as early as 2 min) after detection of a behavioral seizure. These findings suggest that increased pERK labeling could be one of the earliest immunohistochemical indicators of neurons that are activated at the time of a spontaneous seizure.

GABAA receptor alpha 4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol.

The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABAA receptors (GABAA-Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to "tonic" inhibition that can control levels of excitability and network activity. The GABAA-R alpha4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABAA-R-mediated tonic inhibition. Mice were engineered to lack the alpha4 subunit by targeted disruption of the Gabra4 gene. alpha4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABAA-Rs containing the alpha4 subunit and that gaboxadol achieves its effects via the activation of this GABAA-R subtype.

Comparison of three IMRT inverse planning techniques that allow for partial esophagus sparing in patients receiving thoracic radiation therapy for lung cancer.

The purpose of this study is to compare 3 intensity-modulated radiation therapy (IMRT) inverse treatment planning techniques as applied to locally-advanced lung cancer. This study evaluates whether sufficient radiotherapy (RT) dose is given for durable control of tumors while sparing a portion of the esophagus, and whether large number of segments and monitor units are required. We selected 5 cases of locally-advanced lung cancer with large central tumor, abutting the esophagus. To ensure that no more than half of the esophagus circumference at any level received the specified dose limit, it was divided into disk-like sections and dose limits were imposed on each. Two sets of dose objectives were specified for tumor and other critical structures for standard dose RT and for dose escalation RT. Plans were generated using an aperture-based inverse planning (ABIP) technique with the Cimmino algorithm for optimization. Beamlet-based inverse treatment planning was carried out with a commercial simulated annealing package (CORVUS) and with an in-house system that used the Cimmino projection algorithm (CIMM). For 3 of the 5 cases, results met all of the constraints from the 3 techniques for the 2 sets of dose objectives. The CORVUS system without delivery efficiency consideration required the most segments and monitor units. The CIMM system reduced the number while the ABIP techniques showed a further reduction, although for one of the cases, a solution was not readily obtained using the ABIP technique for dose escalation objectives.

Reference dosimetry in clinical high-energy electron beams: comparison of the AAPM TG-51 and AAPM TG-21 dosimetry protocols.

A comparison of the determination of absorbed dose to water in reference conditions with high-energy electron beams (Enominal of 6, 8, 10, 12, 15, and 18 MeV) following the recommendations given in the AAPM TG-51 and in the original TG-21 dosimetry protocols has been made. Six different ionization chamber types have been used, two Farmer-type cylindrical (PTW 30001, PMMA wall; NE 2571, graphite wall) and four plane parallel (PTW Markus, and Scanditronix-Wellhöfer NACP, PPC-05 and Roos PPC-40). Depending upon the cylindrical chamber type used and the beam energy, the doses at dmax determined with TG-51 were higher than with TG-21 by about 1%-3%. Approximately 1% of this difference is due to the differences in the data given in the two protocols; another 1.1%-1.2% difference is due to the change of standards, from air-kerma to absorbed dose to water. For plane-parallel chambers, absorbed doses were determined by using two chamber calibration methods: (i) direct use of the ADCL calibration factors N(60Co)D,w and Nx for each chamber type in the appropriate equations for dose determination recommended by each protocol, and (ii) cross-calibration techniques in a high-energy electron beam, as recommended by TG-21, TG-39, and TG-51. Depending upon the plane-parallel chamber type used and the beam energy, the doses at dmax determined with TG-51 were higher than with TG-21 by about 0.7%-2.9% for the direct calibration procedures and by 0.8%-3.2% for the cross-calibration techniques. Measured values of photon-electron conversion kecal, for the NACP and Markus chambers were found to be 0.3% higher and 1.7% lower than the corresponding values given in TG-51. For the PPC-05 and PPC-40 (Roos) chamber types, the values of kecal were measured to be 0.889 and 0.893, respectively. The uncertainty for the entire calibration chain, starting from the calibration of the ionization chamber in the standards laboratory to the determination of absorbed dose to water in the user beam, has been analyzed for the two formalisms. For cylindrical chambers, the observed differences between the two protocols are within the estimated combined uncertainty of the ratios of absorbed doses for 6 and 8 MeV; however, at higher energies (10< or =E< or =18 MeV), the differences are larger than the estimated combined uncertainties by about 1%. For plane-parallel chambers, the observed differences are within the estimated combined uncertainties for the direct calibration technique; for the cross-calibration technique the differences are within the uncertainty estimates at low energies whereas they are comparable to the uncertainty estimates at higher energies. A detailed analysis of the reasons for the discrepancies is made which includes comparing the formalisms, correction factors, and quantities in the two protocols, as well as the influence of the implementation of the different standards for chamber calibration.

Oblique meniscomeniscal ligament: another potential pitfall for a meniscal tear--anatomic description and appearance at MR imaging in three cases.

Three patients with an arthroscopically proved normal variant, the oblique meniscomeniscal ligament, underwent prospective magnetic resonance (MR) imaging of the knee. In the first case, the ligament was misinterpreted as a displaced flap tear of the posterior horn of the lateral meniscus. In the two subsequent cases, the ligament was identified correctly at MR imaging as the oblique meniscomeniscal ligament.

Up-regulation of GAD65 and GAD67 in remaining hippocampal GABA neurons in a model of temporal lobe epilepsy.

In the pilocarpine model of chronic limbic seizures, subpopulations of glutamic acid decarboxylase (GAD)-containing neurons within the hilus of the dentate gyrus and stratum oriens of the CA1 hippocampal region are vulnerable to seizure-induced damage. However, many gamma-aminobutyric acid (GABA) neurons remain in these and other regions of the hippocampal formation. To determine whether long-term changes occur in the main metabolic pathway responsible for GABA synthesis in remaining GABA neurons, the levels of mRNA and protein labeling for the two forms of GAD (GAD65 and GAD67) were studied in pilocarpine-treated animals that had developed spontaneous seizures. Qualitative and semiquantitative analyses of nonradioactive in situ hybridization experiments demonstrated marked increases in the relative amounts of GAD65 and GAD67 mRNAs in remaining hippocampal GABA neurons. In addition, immunohistochemical studies demonstrated parallel increases in the intensity of terminal labeling for both GAD65 and GAD67 isoforms throughout the hippocampal formation. These increases were most striking for GAD65, the isoform of GAD that is particularly abundant in axon terminals. These findings demonstrate that, in a neuronal network that is capable of generating seizures, both GAD65 and GAD67 are up-regulated at the gene and protein levels in the remaining GABA neurons of the hippocampal formation. This study provides further evidence for the complexity of changes in the GABA system in this model of temporal lobe epilepsy.

Evidence for changing positions of GABA neurons in the developing rat dentate gyrus.

In recent studies, we demonstrated a distinct change in the distribution of glutamate decarboxylase 67 (GAD67) mRNA-containing neurons within the rat dentate gyrus from embryonic day 20 (E20) to postnatal day 15 (PN15) (Dupuy and Houser, J Comp Neurol 1997;389:402-418). We also observed a similar changing pattern for cells with birthdates of many of the mature GAD-containing neurons in the dentate gyrus (Dupuy and Houser, J Comp Neurol 1997;389:402-418). These observations suggested that some early-appearing GABA neurons within the developing molecular layer of the dentate gyrus may gradually alter their positions to become the mature GABAergic cells along the inner border of the granule cell layer. The goal of the present study was to provide additional evidence for our hypothesis by demonstrating the spatial relationships between GAD-containing neurons and granule cells at progressively older ages during development. In this study, immunohistochemical or in situ hybridization methods for the localization of GAD67 or its mRNA were combined with bromodeoxyuridine birthdating techniques that labeled early-generated granule cells with birthdates on E17. At E20, GAD67-containing neurons were located above the granule cell layer that contained E17 birthdated granule cells. During the first two postnatal weeks, both GAD67 mRNA-containing neurons and early-born granule cells were primarily concentrated within the granule cell layer. Double-labeled neurons were rarely observed, and this suggests that these two groups are separate populations. By PN15-PN30, most GAD67 mRNA-containing neurons were distributed along the base of the granule cell layer, significantly below the E17 birthdated granule cells. These findings support our new hypothesis that mature GABA neurons along the inner border of the granule cell layer reach their positions by migrating or translocating through the developing granule cell layer.

Ultrastructural localization of dynorphin in the dentate gyrus in human temporal lobe epilepsy: a study of reorganized mossy fiber synapses.

Substantial reorganization of mossy fibers from granule cells of the dentate gyrus occurs in a high percentage of humans with medically intractable temporal lobe epilepsy. To identify these fibers and determine their ultrastructural features in human surgical specimens, we used preembedding immunoperoxidase labeling of dynorphin A, an opioid peptide that is abundant in normal mossy fibers. In electron microscopic preparations, dynorphin A immunoreactivity was highly associated with dense core vesicles and was localized predominantly in axon terminals in the inner molecular layer of the dentate gyrus, although some dynorphin-labeled dense core vesicles were also observed in dendritic shafts and spines. The labeled terminal profiles were numerous, and, whereas they varied greatly in size, many were relatively large (2.3 microm in mean major diameter). The terminals contained high concentrations of clear round vesicles and numerous mitochondrial profiles, formed distinct asymmetric synapses, often had irregular shapes, and, thus, exhibited many features of normal mossy fiber terminals. The dynorphin-labeled terminals formed synaptic contacts primarily with dendritic spines, and some of these spines were embedded in large labeled terminals, suggesting that they were complex spines. The labeled terminals frequently formed multiple synaptic contacts with their postsynaptic elements, and perforated postsynaptic densities, with and without spinules, were present at some synapses. These findings suggest that the reorganized mossy fiber terminals in humans with temporal lobe epilepsy form abundant functional synapses in the inner molecular layer of the dentate gyrus, and many of these contacts have ultrastructural features that could be associated with highly efficacious synapses.

Children's responses to immunizations: lullabies as a distraction.

The purpose of this study was to investigate the effects of audiotaped lullabies on physiological and behavioral distress and perceived pain among children during routine immunization. An experimental design was used to study 99 healthy children ages 3 to 6 years. Half the children received the musical intervention during the immunizations, while the other half did not. Groups were assessed during five phases: baseline, preimmunizations, during the immunization, after Band-Aid application, and 2 min after phase 4. Physiological variables (heart rate, blood pressure) were obtained in phases 1, 4, and 5. Behavioral distress was measured using the Observational Scale of Behavioral Distress during phases 1, 2, 3, and 4. Pain perception was measured using the Oucher in phases 1 and 4. No significant differences were found between experimental and control groups for heart rate, blood pressure, or Oucher scores. However, total distress scores were significantly less for the experimental group. These results indicate that immunization is a stressful experience for children. Recommendations include further study incorporating pharmacological and nonpharmacological interventions.

Developmental changes in GABA neurons of the rat dentate gyrus: an in situ hybridization and birthdating study.

The temporal and spatial distribution of glutamate decarboxylase 67 (GAD67) mRNA-containing neurons in the dentate gyrus was analyzed from embryonic day 20 (E20) to postnatal day 15 (PN15) with nonradioactive in situ hybridization methods. A major goal was to follow the development of an early-appearing population of gamma-aminobutyric acid (GABA) neurons within the developing molecular layer. At E20, GAD67 mRNA-containing neurons were highly concentrated slightly above the outer border of the developing granule cell layer. By PN3-PN5, many labeled cells were distributed within the developing granule cell layer; by PN15, labeled neurons occupied positions normally seen in the adult, such as along the inner border of the granule cell layer. This developmental pattern is unique and led to additional studies to determine the potential fate of the early-appearing GABA population. The possibility of apoptotic cell death was investigated with in situ end labeling techniques at developmental ages E21-PN15. Very few apoptotic cells were detected in the developing molecular layer at all ages examined. Birthdating studies of neurons labeled with bromodeoxyuridine revealed a changing pattern, similar to that of GAD67 mRNA, for cells with birthdates (E14) of many of the mature GAD-containing neurons in the dentate gyrus. The changes in the mRNA and birthdating patterns from E20-PN15 suggest that some of the early-appearing GABA neurons within the developing molecular layer of the dentate gyrus may alter their positions to eventually become the mature GABA population along the inner border of the granule cell layer.

Vulnerability and plasticity of the GABA system in the pilocarpine model of spontaneous recurrent seizures.

Several similarities exist between the alterations observed in the chronic pilocarpine model of recurrent seizures in the rat and those found in human temporal lobe epilepsy. The present studies are focused on changes in the GABA system in this model. Following the initial pilocarpine-induced seizures, a substantial loss of glutamic acid decarboxylase (GAD) mRNA-containing neurons has been found in the hilus of the dentate gyrus (Obenaus et al., J. Neurosci., 13 (1993) 4470-4485), and, recently, a loss of GAD mRNA-labeled neurons has also been found in stratum oriens of CA1. Yet numerous other GABA neurons remain within the hippocampal formation, and there appear to be multiple compensatory changes in these neurons. Labeling for GAD65 mRNA and associated protein is substantially increased in the remaining GABA neurons at 2-4 months after the initial seizure episode. Such increased labeling suggests that the remaining GABA neurons are part of a functional circuit and may be responding to the need for increased activity. Alterations also occur in at least one subunit of the GABA-A receptor. Labeling for the alpha(5) subunit mRNA is substantially decreased in CA1 and CA2 of pilocarpine-treated rats during the chronic, seizure-prone period. These findings emphasize the complexity of changes in the GABA system and indicate a need for evaluating the functional consequences of each of the changes. The initial loss of specific groups of GABA neurons could be a critical first step in the gradual development of epileptiform activity. While many of the subsequent changes in the GABA system may be considered to be compensatory, significant deficits of GABAergic function could remain.

Prominent expression of two forms of glutamate decarboxylase in the embryonic and early postnatal rat hippocampal formation.

Immunohistochemical methods were used to determine the earliest times of detection for two forms of glutamate decarboxylase (GAD67 and GAD65) in the embryonic and early postnatal rat hippocampal formation and to determine whether their distribution patterns differed from each other and from those of the adult. Both GAD67- and GAD65-containing neurons were observed as early as embryonic day 17 (E17)-E18 in the hippocampus and E19 in the dentate gyrus, and this was substantially earlier than GAD had been detected previously in the hippocampal formation. The two GAD isoforms displayed very similar distribution patterns, but these patterns were distinctly different from those of the adult. From E17 to E20, GAD67 and GAD65 were expressed in neuronal cell bodies throughout the hippocampal and dentate marginal zones (future dendritic layers), and relatively few existed within the principal cell body layers, where GAD-positive neurons are frequently concentrated in the adult. At E21 to postnatal day 1 (P1), there was a sudden shift from a predominance of GAD-containing cell bodies within the developing dendritic regions to a meshwork of GAD-positive processes with terminal-like varicosities in these same regions. This pattern also contrasted with that of the adult, in which GAD-labeled terminals are highly concentrated in the principal cell layers. Electron microscopic observations of the GAD-labeled processes at P1 confirmed their axon-like appearance and demonstrated that the immunoreactivity was consistently localized in vesicle-filled regions that were often closely apposed to and, in some instances, established synaptic contacts with dendritic profiles. The present identification of an early abundance of GAD-containing structures in the hippocampal formation and the marked change in their distribution during development complement recent observations of developmental changes in the functioning of the GABA system and provide additional support for the early involvement of this neurotransmitter system in hippocampal development.

Subpopulations of GABA neurons in the dentate gyrus express high levels of the alpha 1 subunit of the GABAA receptor.

The alpha 1 subunit of the gamma-aminobutyric acid (GABA)A receptor is highly expressed in a subgroup of neurons in the hippocampal formation. The distribution and chemical identities of these neurons in the dentate gyrus have been studied with double-labeling in situ hybridization and immunohistochemical methods. Double labeling for the alpha 1 subunit and glutamate decarboxylase 65 (GAD65) mRNAs indicated that virtually all neurons in the dentate gyrus that are heavily labeled for the alpha 1 subunit are GABA neurons. However, many GAD65 mRNA-labeled neurons in the hilus do not contain high levels of the alpha 1 subunit mRNA and protein. Studies were thus conducted to determine if the somatostatin neurons of the hilus were part of the alpha 1 subunit-labeled group. Double labeling for the alpha 1 subunit and pre-prosomatostatin mRNAs demonstrated virtually no co-localization of these mRNAs in hilar neurons. Thus, the strongly labeled alpha 1 mRNA-containing neurons and the somatostatin neurons constitute two distinct populations of hilar GABA neurons. Double labeling for the alpha 1 subunit polypeptide and its mRNA with immunohistochemical and in situ hybridization methods demonstrated directly that neurons of the dentate gyrus that express high levels of the alpha 1 subunit mRNA are the same neurons that show extensive labeling for the alpha 1 subunit along their somal and dendritic surfaces. The high levels of alpha 1 subunit expression in some populations of GABA neurons could be related to prominent disinhibitory functions of these neurons.

Experimentally induced disorders of neuronal migration produce an increased propensity for electrographic seizures in rats.

Disorders of neuronal migration in humans are associated with intractable epilepsy and some evidence suggests a causal relationship. This study evaluated electroencephalograms (EEG) of rats with experimentally induced disorders of neuronal migration. Fetal Sprague-Dawley rats were exposed to 196 cGy external irradiation on days 16 and 17 of gestation. This produced adult offspring with diffuse cortical dysplasias, agenesis of the corpus callosum, periventricular heterotopias, and dispersion of the pyramidal cell layer of the hippocampus. Epidural electrodes were implanted in four experimental (irradiated on gestational day 17) and four control rats. EEGs were recorded without anesthesia and in the presence of the anesthetic agents ketamine, acepromazine, and xylazine. In the presence of acepromazine, xylazine, or a combination of the two drugs, two of the four experimental rats had prolonged ictal activity on EEG. In one of the rats the ictal activity progressed to electrographic status epilepticus. Ketamine alone did not produce ictal EEG activity. None of the control rats demonstrated ictal activity under any treatment condition. This study demonstrates that disorders of neuronal migration are associated with an increased propensity for seizures in the presence of certain sedating agents.

Somatostatin neurons are a subpopulation of GABA neurons in the rat dentate gyrus: evidence from colocalization of pre-prosomatostatin and glutamate decarboxylase messenger RNAs.

The distribution and extent of glutamate decarboxylase 65 (GAD65) mRNA-labeled neurons that coexpress pre-prosomatostatin mRNA were studied in the rat dentate gyrus of the dorsal and ventral hippocampal formation. The distribution of each group of neurons was determined initially by nonradioactive in situ hybridization experiments with digoxigenin-labeled riboprobes for GAD65 mRNA and pre-prosomatostatin mRNA. Double labeling experiments were then conducted with digoxigenin-labeled riboprobes for GAD65 mRNA and 35S-labeled riboprobes for pre-prosomatostatin mRNA. In the dorsal and ventral dentate gyrus, GAD65 mRNA-containing neurons were highly concentrated in the hilus and in the innermost part of the granule cell layer whereas only a few labeled neurons were scattered in the molecular layer. Pre-prosomatostatin mRNA-containing neurons were primarily located in the hilus and were virtually absent from the molecular and granule cell layers. The simultaneous detection of GAD65 and pre-prosomatostatin mRNAs in the same sections showed that the vast majority of pre-prosomatostatin mRNA-containing neurons in the hilus of the dentate gyrus were also labeled for GAD65 mRNA. In contrast many GAD65 mRNA-labeled neurons did not contain pre-prosomatostatin mRNA. These included all neurons in the molecular layer, neurons within the inner granule cell layer and neurons interspersed amongst double labeled neurons in the hilus. Quantitative analyses indicated that a very high percentage of hilar pre-prosomatostatin mRNA-containing neurons coexpressed GAD65 mRNA in the dorsal (96%) and ventral (92%) dentate gyrus. In contrast only a part of the total population of hilar GAD65 mRNA-containing neurons were also labeled for pre-prosomatostatin mRNA in the dorsal (43%) and ventral (53%) dentate gyrus. In the CA3c region, the percentages of neurons containing both mRNAs were similar to those observed in the hilus. The findings demonstrate that the vast majority of hilar somatostatin neurons, which have previously been shown to be extremely vulnerable to ischemia and seizure-induced damage, are GABA neurons. However, the total population of GAD65 mRNA-containing neurons in the hilus is substantially larger than the somatostatin-containing subgroup, and these findings reinforce the suggestion that GABA neurons are a major component of the diverse group of neurons in the hilus of the dentate gyrus.