A benchmark for Monte Carlo simulations in gamma-ray spectrometry Part II: True coincidence summing correction factors.

The goal of this study is to provide a benchmark for the use of Monte Carlo simulation when applied to coincidence summing corrections. The examples are based on simple geometries: two types of germanium detectors and four kinds of sources, to mimic eight typical measurement conditions. The coincidence corrective factors are computed for four radionuclides. The exercise input files and calculation results with practical recommendations are made available for new users on a dedicated webpage.

Moduladores cronobiológicos en la efectividad de la trombolisis prehospitalaria / Circadian modulation of effectiveness in prehospital thrombolysis

Objetivo: Investigar si la hora del día influye en la efectividad de la terapia trombolítica prehospitalaria en pacientes con infarto agudo de miocardio con elevación del segmento ST (IAMCEST). Método: Estudio observacional de cohortes retrospectivo con pacientes diagnosticados de IAMCEST a quienes se les realizó trombolisis precoz prehospitalaria. Se analizaron, como variables predictoras independientes de la efectividad de la terapia trombolítica, la hora del día de administración de la terapia trombolítica (variable principal), divididos en periodos horarios de 6 h y de 12 h, la edad, el sexo, la hora inicio del dolor torácico, tiempo de evolución del infarto, los factores de riesgo cardiovascular y el área de localización del infarto. Los datos se obtuvieron de la historia clínica y del seguimiento de preavisos hospitalarios a las 24 h. Resultados: Se incluyó a 206 pacientes. Dos variables se muestran como predictores independientes de la efectividad de la trombolisis prehospitalaria: la hora del día de administración de la terapia trombolítica, en el rango de cronorriesgo cardiovascular de 6a 12 h, con respecto al resto de franjas horarias (0-6 h, 12-18 h, 18-24 h) [p = 0,005odds ratio (OR) = 2,46; intervalo de confianza (IC) del 95%, 1,30-4,64] y presentar cardiopatía isquémica previa) (p = 0,003, OR = 5,30; IC del 95%, 1,74-16,15).Conclusiones: Encontramos variaciones circadianas clínicamente significativas en la efectividad del tratamiento trombolítico prehospitalario administrado a los pacientes con IAMCEST, independientemente del agente trombolítico empleado, de manera que existe una tromborresistencia matinal (6-12 am) al tratamiento y una mayor efectividad de reperfusión coronaria cuando se administra en el resto de franjas horarias diurnas ,especialmente en la de tarde (12-18 h) (AU)

Objective: To study whether time of day influences the effectiveness of prehospital thrombolysis in patients who have had acute myocardial infarction with ST-segment elevation (STEMI).Methods: Observational study of retrospective cohorts. We included patients diagnosed with STEMI who received early application of prehospital thrombolytic therapy. The main variable studied as an independent predictor of effectiveness was the time of day the thrombolytic agent was administered; this variable was studied in 6-hour periods and 12-hourperiods. Additional independent variables were patient age and sex, onset of chest pain, duration of pain from onset until administration of the thrombolytic agent, cardiovascular risk factors, and location of infarction. Data were extracted from patient records and ambulance pre-alert calls in the next 24 hours. Results: Two hundred six patients were studied. The 2 independent variables that predicted the effectiveness of prehospital thrombolysis were administration of the thrombolytic agent at a time of day within the period of greatest cardiovascular risk (6 AM to 12 noon) in comparison with the other time frames (12 midnight to 6 AM, 12 noon to 6 PM and 6 PM to midnight) (odds ratio [OR], 2.46; 95% CI, 1.30-4.64; P=.005) and history of ischemic heart disease (OR,5.30; 95% CI, 1.74-16.15; P=.003).Conclusions: We found that circadian rhythm had a clinically significant effect on the effectiveness of prehospital thrombolysis in STEMI patients. The effect was present regardless of which thrombolytic agent was used. The greatest resistance to therapy was observed in the morning hours between 6 AM and 12 noon. The response was greater in the remaining time frames and greatest in the hours between noon and 6 PM (AU)

Monaural conductive hearing loss alters the expression of the GluA3 AMPA and glycine receptor α1 subunits in bushy and fusiform cells of the cochlear nucleus.

The impact of conductive hearing loss (CHL), the second most common form of hearing loss, on neuronal plasticity in the central auditory pathway is unknown. After short-term (1 day) monaural earplugging, the GluA3 subunits of the AMPA receptor (AMPAR) are upregulated at auditory nerve synapses on the projection neurons of the cochlear nucleus; glycine receptor α1 (GlyRα1) subunits are downregulated at inhibitory synapses in the same neuronal population. These data suggest that CHL affects receptor trafficking at synapses. We examined the impact of 7 days of CHL on the general expression of excitatory and inhibitory receptors by quantitative biochemistry and immunohistochemistry, using specific antibodies to detect AMPAR subunits (GluA1, GluA2, GluA2/3, and GluA4), GlyRα1, and the GABA(A) receptor subunits ß2/3. Following monaural earplugging and an elevation of the hearing threshold by approximately 35 dB, the immunolabeling of the antibody for the GluA2/3 subunits but not the GluA2 subunit increased on bushy cells (BCs) and fusiform cells (FCs) of the ipsilateral ventral and dorsal cochlear nuclei. These same cell types showed a downregulation of the GlyRα1 subunit. Similar results were observed in the contralateral nuclei. The expression levels of GABA(A) ß2/3 were unchanged. These findings suggest that, following longer periods of monaural conductive hearing loss, the synthesis and subsequent composition of specific glutamate and glycine receptors in projection neurons and their synapses are altered; these changes may contribute to abnormal auditory processing.

Clustered and non-clustered GABAA receptors in cultured hippocampal neurons.

In cultured hippocampal neurons, gamma2 subunit-containing GABA(A) Rs form large postsynaptic clusters at GABAergic synapses and small clusters outside GABAergic synapses. We now show that a pool of non-clustered gamma2 subunit-containing GABA(A) Rs are also present at the cell surface. We also demonstrate that myc- or EGFP-tagged gamma2, alpha2, beta3 or alpha1 subunits expressed in these neurons assemble with endogenous subunits, forming GABA(A) Rs that target large postsynaptic clusters, small clusters outside GABAergic synapses or a pool of non-clustered surface GABA(A) Rs. In contrast, myc- or EGFP-tagged delta subunits only form non-clustered GABA(A) Rs, which can be induced to form clusters by antibody capping. A myc-tagged chimeric gamma2 subunit possessing the large intracellular loop (IL) of the delta-subunit IL (myc gamma2S/delta-IL) assembled into GABA(A) Rs, but it did not form clusters, therefore behaving like the delta subunit. Thus, the large intracellular loops of gamma2 and delta play an important role in determining the synaptic clustering/non-clustering capacity of the GABA(A) Rs.

[Metastatic bone disease. Diagnosis and treatment]. / Enfermedad metastásica ósea. Diagnóstico y tratamiento.

The high incidence of bone metastasis secondary to carcinomas and its serious functional repercussion are motives for constant study and advance in the methods of evaluation, diagnosis and treatment. Pain is the most frequently shown symptom, although at times the start is a pathological fracture. The classic tests of detection and evaluation of the spread of the metastatic disease, simple radiology and gammagraphy, are today complemented by others such as computerised tomography (CT) and magnetic resonance (MR), improving the information on the characteristics of the lesion both inside and outside the bone. On the other hand, positron emission tomography (PET) is showing a far higher sensitivity than gammagraphy and will probably be the test of the future for the early detection of metastasis and of silent primary tumours. The possibilities of treatment of bone metastasis are based on the use of bone regenerators, radiotherapy and surgery. The former two are indicated in lesions already detected in radiography, whether symptomatic or not, if there is no foreseeable risk of fracture. Surgery is indicated in situations of poor or null response to those treatments, when the risk of fracture is high or a pathological fracture has been produced. Before any therapeutic planning, a detailed evaluation of the patient must be carried out, both at a local level (size, site, extension of the metastasis) and general (type of primary tumour, phase of treatment and response, estimated survival).

[Cystic pancreatic tumours and pseudo-tumoural lesions]. / Tumores quísticos pancreáticos y lesiones pseudotumorales.

Cystic lesions of the pancreas are infrequent, estimated at only some 1% of all pancreatic tumours and at some 10% of all pancreatic cysts. The pre-operational diagnosis is important for a suitable treatment, with valuable radiological techniques available today such as ultrasound, computerised tomography and magnetic resonance. In spite of this we have to accept that we are facing a group of tumours whose diagnosis is difficult, due to the great variety of cellular types existing within them.

Biochemical analysis of GABA(A) receptor subunits alpha 1, alpha 5, beta 1, beta 2 in the hippocampus of patients with Alzheimer's disease neuropathology.

Alzheimer's disease (AD) is characterized by selective vulnerability of specific neuronal populations within particular brain regions. For example, hippocampal glutamatergic cell populations within the CA1/subicular pyramidal cell fields have been found to be particularly vulnerable early in AD progression. In contrast, hippocampal GABA-ergic neurons and receptors appear resistant to neurodegeneration. Despite relative sparing of GABA(A) receptors in AD, it is possible that the specific subunit composition of these receptors may undergo alterations with disease progression. In order to address this issue, we employed quantitative Western blot analysis to examine protein levels of GABA(A) receptor subunits alpha 1, alpha 5, beta 1, beta 2 in the hippocampus of subjects displaying increasing severity of AD neuropathology. Subjects were categorized into three groups based upon Braak staging pathologic criteria: pathologically mild (stages I/II, n=9); moderate (stages III/IV, n=8); and severe (stages V/VI, n=7). Across all subject groups, levels of subunit protein were heterogeneously distributed throughout the five hippocampal subregions analyzed (subiculum, CA1-3, dentate gyrus). Statistical analyses revealed differential preservation of GABA(A) receptor subunits in AD. In particular, alpha 1, beta 1, and beta 2 displayed little difference in protein levels among pathologically mild, moderate, and severe subject groups. In contrast, although relatively modest, protein levels of the alpha 5 subunit were significantly reduced between subjects with severe neuropathology compared with pathologically mild subjects (13.5% reduction). Collectively, our data provide evidence for heterogeneous distribution and relative sparing of GABA(A) receptor subunits in the hippocampus of AD patients.

Immunocytochemical localization of the beta(3) subunit of the gamma-aminobutyric acid(A) receptor in the rat brain.

A novel anti-beta(3) subunit-specific GABA(A) receptor (GABA(A)R) antibody has been prepared by immunizing a rabbit with a bacterial fusion protein of the large intracellular loop of the beta(3) subunit. The antiserum immunoprecipitated the solubilized GABA(A) receptor. The anti-beta(3) antibody was affinity purified on immobilized beta(3) large intracellular loop peptide. In immunoblots, the purified antibody reacted with a 57 KDa peptide. Immunocytochemistry with the affinity-purified antibody has revealed the localization of the beta(3) subunit in the rat brain. A comparative study with the immunocytochemical distribution of the beta(2) subunit has also been performed. There are areas of the brain and cell types where the distribution of beta(2) and beta(3) overlap (i.e., cerebral cortex, cerebellum,and most layers of the olfactory bulb). There are also clear differences in the expression of beta(3) and beta(2) in other brain areas and cell types. Thus, high beta(3) but low or no beta(2) expression was observed in the corpus striatum and in granule cells of the olfactory bulb. In the hippocampus the expression of beta(3) was considerably higher than that of beta(2), but some hippocampal interneurons showed high expression of beta(2). High beta(2) but little or no expression of beta(3) was observed in thalamic nuclei, substantia nigra, globus pallidus, inferior colliculus and the short axon cells of the olfactory bulb.

Normal electrophysiological and behavioral responses to ethanol in mice lacking the long splice variant of the gamma2 subunit of the gamma-aminobutyrate type A receptor.

The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.

Dopamine D2 receptor effects on GABA(A) receptor expression may modify melanotrope peptide release.

Stimulation of melanotrope dopamine D2 receptors decreases mitotic rate, calcium channel activity, and the biosynthesis of several proteins. This study demonstrates that D2 receptor activation also affects GABA(A) receptor beta2/beta3 subunit immunoreactivity. Following chronic treatment with haloperidol, a D2 receptor antagonist, GABA(A) receptor immunoreactivity increased, whereas it decreased after chronic treatment with bromocriptine, a dopamine D2 receptor agonist. Thus, these data indicate that D2 function regulates GABA(A) receptor expression in melanotropes, a mechanism by which peptide release may be modified.

N-acetylaspartylglutamate stimulates metabotropic glutamate receptor 3 to regulate expression of the GABA(A) alpha6 subunit in cerebellar granule cells.

We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 microM) results in the transient increase in content of GABA(A) alpha6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl) glycine and (2S)-alpha-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in alpha6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, alpha2-adrenergic, muscarinic, and GABA(B) receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in alpha6 levels induced by 30 microM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the beta-adrenergic receptor agonist isoproterenol. The increase in alpha6 mRNA content is followed by a fourfold increase in alpha6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional alpha6-containing GABA(A) receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA(A) alpha6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA(A) receptor subunit composition.

Coexistence of two beta subunit isoforms in the same gamma-aminobutyric acid type A receptor.

Three novel subunit-specific antisera to the beta1, beta2, and beta3 subunits of rat gamma-aminobutyric acid type A (GABAA) receptors have been used to study the native receptor in the rat brain. Affinity-purified anti-beta1, anti-beta2, and anti-beta3 antibodies recognized in immunoblots protein bands of 57, 55, and 57 kDa, respectively. Quantitative immunoprecipitation of solubilized GABAA receptors from various rat brain regions showed that the beta2 subunit was the most abundant isoform in cerebellum (in 96% of the GABAA receptors) and cerebral cortex (64%) but that it was the least abundant isoform in hippocampus (44%). The beta3 subunit was found most abundant in hippocampus (64%) followed by cerebral cortex (48%) and cerebellum (33%). The beta1 subunit was present in a very small proportion of the cerebellar GABAA receptors (3%), but it was present in a high proportion of the GABAA receptors from the hippocampus (49%) and cerebral cortex (32%). Quantitative receptor immunoprecipitation or immunopurification followed by immunoblotting experiments have revealed the existence of colocalization of two different beta subunit isoforms in a significant proportion of the brain GABAA receptors. Thus, in the rat cerebral cortex 33% of the GABAA receptors have both beta2 and beta3 subunits, and 19% of the receptors have both beta1 and beta3 subunits. The extent of colocalization of beta subunit isoforms varied among brain regions, being highest in hippocampus and lowest in cerebellum. These and other results taken together suggest that the number of alpha, beta, and gamma subunits (stoichiometry) in the brain GABAA receptor pentamers might not be unique. It might vary depending on receptor type.

GABAA receptor subunit expression changes in the rat cerebellum and cerebral cortex during aging.

Significant aging-related decreased expression of various GABAAR subunit mRNAs (alpha 1, gamma 2, beta 2, beta 3 and sigma) was found in both cerebellum and cerebral cortex using quantitative dot blot and in situ hybridization techniques. Contrary to the other subunits, the alpha 6 mRNA expression was significantly increased in the aged cerebellum. Parallel age-related changes in protein expression for gamma 2 and beta 2/3 (decrease) and alpha 6 (increase) were revealed in cerebellum by quantitative immunocytochemistry. However, no significant changes in alpha 1 protein expression nor in the number or affinity of [3H]zolpidem binding sites were detected in cerebellum even though alpha 1 mRNA expression was significantly decreased in the aged rat. Age-related increased expression of alpha 6 mRNA and protein in the cerebellum was accompanied by no significant changes in the number of diazepam-insensitive [3H]Ro15-4513 binding sites. In the cerebral cortex, no changes in the protein expression of the main GABAA receptor subunits (alpha 1, gamma 2 and beta 2/3) were observed which contrasted with the age-related decreased expression of the corresponding mRNAs. No significant changes in the number or affinity of [3H]zolpidem binding sites were observed in the cerebral cortex. Thus, age-related changes in the mRNA expression of a particular subunit does not necessarily lead to similar changes in protein or assembly into mature GABAA receptors. The results reveal the existence of complex regulatory mechanisms of GABAA receptor expression, at the transcriptional, translational and post-translational and/or assembly levels, which vary with the subunit and brain area.

Aging-related subunit expression changes of the GABAA receptor in the rat hippocampus.

Aging-related changes in the subunit expression of some hippocampal GABAA receptors have been found. Quantitative in situ hybridization has revealed that alpha 1, subunit messenger RNA expression was significantly increased in the hippocampus (34%) of old rats. The largest increases were observed in the dentate gyrus (76%) and in the CA1 field (30%). Quantitative immunocytochemistry also showed increased protein expression of the alpha 1 subunit in the dentate gyrus (19%) and CA1 (14%) of old rats. The increased alpha 1 messenger RNA and protein expression led to increased proportions of assembled GABAA receptors that contained alpha 1 subunits, as revealed by quantitative immunoprecipitation of (3H)flunitrazepam and (3H)muscimol binding. In contrast, there were no significant changes in the expression of beta 2, beta 3 and total gamma 2 (gamma 2S + gamma 2L) subunits, although a slightly increased expression of gamma 2L peptide was detected in the hippocampus proper (7%), but not in the dentate gyrus. The results are consistent with the notion that in the rat hippocampus there is an aging-related change in the subunit composition of some GABAA receptors.

Distribution of immunoreactivity for the beta 2 and beta 3 subunits of the GABAA receptor in the mammalian spinal cord.

The localization of GABAA receptors in cat and rat spinal cord was analyzed using two monoclonal antibodies specific for an epitope shared by the beta 2 and beta 3 subunits of the receptor. beta 2/beta 3-subunit immunoreactivity was the most intense in inner lamina II, lamina III, and lamina X, and it was the least intense in lamina IX. In laminae I-III, generally, the staining had a rather diffuse appearance, but the surfaces of small cell bodies in these laminae were outlined clearly by discrete labeling, as were many cell bodies and dendrites in deeper laminae. Rhizotomy experiments and ultrastructural observations indicated that beta 2/beta 3-subunit immunoreactivity in the dorsal horn was largely localized in intrinsic neuropil elements rather than in the terminals of primary afferent fibers, even though labeling overlapped with the terminal fields of different types of primary afferents and was also detected on the membranes of dorsal root ganglion neurons. With few exceptions (most notably, a highly immunoreactive group of dorsolaterally located cells in the cat lumbar ventral horn), motoneurons expressed low levels of beta 2/beta 3-subunit immunoreactivity. Labeling of neuronal membranes was fairly continuous, but focal accumulations of beta 2/beta 3-subunit immunoreactivity were also detected using immunofluorescence. Focal "hot spots" correlated ultrastructurally with the presence of synaptic junctions. Dual-color immunofluorescence revealed that focal accumulations of beta 2/beta 3-subunit immunoreactivity were frequently apposed by glutamic acid decarboxylase (GAD)-immunoreactive terminals. However, the density of continuous-membrane beta 2/beta 3 immunolabeling and GAD terminal density were not correlated in many individual neurons. The results suggest the existence of "classical" (synaptic) and "nonclassical" (paracrine) actions mediated via spinal cord GABAA receptors. The study also revealed the relative paucity of beta 2/beta 3-subunit immunoreactivity postsynaptic to certain GABAergic terminals, particularly those presynaptic to motoneurons or primary afferent terminals.

Immunocytochemical localization of the alpha 6 subunit of the gamma-aminobutyric acidA receptor in the rat nervous system.

The localization in the rat central nervous system and retina of the alpha 6 subunit peptide of the gamma-aminobutyric acid (GABAA) receptor has been studied by light microscopy immunocytochemistry with a specific anti-alpha 6 antibody. The alpha 6 subunit was present in the granule cells of the cerebellum, the granule cells of the dorsal cochlear nucleus, axons of the olfactory nerve including the glomerular endings, layer II of the dorsal horn of the spinal cord, and in the retinal synaptic layers, particularly the inner plexiform layer. Thus, contrary to the general belief, the alpha 6 subunit is not exclusively localized in the granule cells of the cerebellum. It is also expressed in some sensory neurons and other neurons involved in the initial processing of sensory information.

Brain GABAA receptors studied with subunit-specific antibodies.

Brain GABAA/benzodiazepine receptors are highly heterogeneous. This heterogeneity is largely derived from the existence of many pentameric combinations of at least 16 different subunits that are differentially expressed in various brain regions and cell types. This molecular heterogeneity leads to binding differences for various ligands, such as GABA agonists and antagonists, benzodiazepine agonists, antagonists, and inverse agonists, steroids, barbiturates, ethanol, and Cl- channel blockers. Different subunit composition also leads to heterogeneity in the properties of the Cl- channel (such as conductance and open time); the allosteric interactions among subunits; and signal transduction efficacy between ligand binding and Cl- channel opening. The study of recombinant receptors expressed in heterologous systems has been very useful for understanding the functional roles of the different GABAA receptor subunits and the relationships between subunit composition, ligand binding, and Cl- channel properties. Nevertheless, little is known about the complete subunit composition of the native GABAA receptors expressed in various brain regions and cell types. Several laboratories, including ours, are using subunit-specific antibodies for dissecting the heterogeneity and subunit composition of native (no reconstituted) brain GABAA receptors and for revealing the cellular and subcellular distribution of these subunits in the nervous system. These studies are also aimed at understanding the ligand-binding, transduction mechanisms, and channel properties of the various brain GABAA receptors in relation to synaptic mechanisms and brain function. These studies could be relevant for the discovery and design of new drugs that are selective for some GABAA receptors and that have fewer side effects.

The gamma subunits of the native GABAA/benzodiazepine receptors.

Subunit-specific antibodies to all the gamma subunit isoforms described in mammalian brain (gamma(1), gamma(2S), gamma(2L), and gamma(3) have been made. The proportion of GABA(A) receptors containing each gamma subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the gamma(1) and gamma(3) subunits are present in considerable smaller proportion of GABA(A) receptor than the gamma(2) subunit. Immunocytochemistry shows that gamma(1) immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, gamma(1) immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, gamma(3) immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in gamma(2S). In the cerebellum, gamma(1)++ immunolabeling was localized on the Bergmann glia. The gamma(2S) and gamma(2L) subunits are differentially expressed in various brain regions. Thus the gamma(2S) is highly expressed in the olfactory bulb and hippocampus whereas the gamma(2L) is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The gamma(2S) and gamma(2L) coexist in some brain areas and cell types. Moreover, the gamma(2S) and gamma(2L) subunits can coexist in the same GABA(A) receptor pentamer. We have shown that this is the case in some GABA(A) receptors expressed in cerebellar granule cells. These GABA(A) receptors also have alpha and beta subunits forming the pentamer. Immunoblots have shown that the rat gamma(1), gamma(2S), gamma(2L) and gamma(3) subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the gamma(2S) and gamma(2L) subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA(A) receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various gamma subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA(A) receptors in brain.