Time domain diffuse Raman spectrometer based on a TCSPC camera for the depth analysis of diffusive media.

We present a time domain diffuse Raman spectrometer for depth probing of highly scattering media. The system is based on, to the best of our knowledge, a novel time-correlated single-photon counting (TCSPC) camera that simultaneously acquires both spectral and temporal information of Raman photons. A dedicated non-contact probe was built, and time domain Raman measurements were performed on a tissue mimicking bilayer phantom. The fluorescence contamination of the Raman signal was eliminated by early time gating (0-212 ps) the Raman photons. Depth sensitivity is achieved by time gating Raman photons at different delays with a gate width of 106 ps. Importantly, the time domain can provide time-dependent depth sensitivity leading to a high contrast between two layers of Raman signal. As a result, an enhancement factor of 2170 was found for our bilayer phantom which is much higher than the values obtained by spatial offset Raman spectroscopy (SORS), frequency offset Raman spectroscopy (FORS), or hybrid FORS-SORS on a similar phantom.

Astrocyte-to-neuron communication through integrin-engaged Thy-1/CBP/Csk/Src complex triggers neurite retraction via the RhoA/ROCK pathway.

Two key proteins for cellular communication between astrocytes and neurons are αvß3 integrin and the receptor Thy-1. Binding of these molecules in the same (cis) or on adjacent (trans) cellular membranes induces Thy-1 clustering, triggering actin cytoskeleton remodeling. Molecular events that could explain how the Thy-1-αvß3 integrin interaction signals have only been studied separately in different cell types, and the detailed transcellular communication and signal transduction pathways involved in neuronal cytoskeleton remodeling remain unresolved. Using biochemical and genetic approaches, single-molecule tracking, and high-resolution nanoscopy, we provide evidence that upon binding to αvß3 integrin, Thy-1 mobility decreased while Thy-1 nanocluster size increased. This occurred concomitantly with inactivation and exclusion of the non-receptor tyrosine kinase Src from the Thy-1/C-terminal Src kinase (Csk)-binding protein (CBP)/Csk complex. The Src inactivation decreased the p190Rho GTPase activating protein phosphorylation, promoting RhoA activation, cofilin, and myosin light chain II phosphorylation and, consequently, neurite shortening. Finally, silencing the adaptor CBP demonstrated that this protein was a key transducer in the Thy-1 signaling cascade. In conclusion, these data support the hypothesis that the Thy-1-CBP-Csk-Src-RhoA-ROCK axis transmitted signals from astrocytic integrin-engaged Thy-1 (trans) to the neuronal actin cytoskeleton. Importantly, the ß3 integrin in neurons (cis) was not found to be crucial for neurite shortening. This is the first study to detail the signaling pathway triggered by αvß3, the endogenous Thy-1 ligand, highlighting the role of membrane-bound integrins as trans acting ligands in astrocyte-neuron communication.

The potent non-competitive mGlu1 receptor antagonist BAY 36-7620 differentially affects synaptic plasticity in area cornu ammonis 1 of rat hippocampal slices and impairs acquisition in the water maze task in mice.

In this study we evaluated the effects of the novel, potent non-competitive metabotropic glutamate receptor (mGluR) 1 antagonist (3aS,6aS)-6a-naphthalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on (BAY 36-7620) on different types of synaptic plasticity in the hippocampal cornu ammonis (CA) 1-region and on hippocampus-dependent spatial learning. After having confirmed the presence of mGluR1 in the hippocampal CA1 region of our rat strain by confocal microscopy, we tested the effects of BAY 36-7620 on: 1) long-term potentiation (LTP) induced by weak and strong stimulation; 2) 3,5-dihydroxyphenylglycine (DHPG, 30 microM)-induced depression of synaptic transmission; and 3) learning of the hidden platform version of the water maze by mice. BAY 36-7620 (10 microM) amplified LTP but, like the mGluR1 antagonists 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt, 10 microM) and 4-carboxyphenylglycine (4-CPG, 50 microM), diminished LTP at 1 microM. The mGluR5 antagonist 6-methyl-2-(phenylethynyl)-pyridine (MPEP, 10 microM) had no effect. BAY 36-7620 (10 microM) did not affect strong LTP. Thus, mGlu 1, but not mGlu 5, receptors modulate LTP elicited by weak stimulation in vitro. DHPG-induced depression of synaptic transmission was only marginally affected by BAY 36-7620 (1 microM) or 4-CPG (100 microM). In a mouse water maze study, BAY 36-7620 (10 mg/kg, i.v.) increased the escape latency and impaired water escape task acquisition during the first 4 days. Drug- and vehicle-treated groups showed comparable performance at day 5. Our data support a role for mGluR1 in LTP and in the acquisition of spatial memory.

Enhanced synaptic excitation-inhibition ratio in hippocampal interneurons of rats with temporal lobe epilepsy.

A common feature of all epileptic syndromes is the repetitive occurrence of pathological patterns of synchronous neuronal activity, usually combined with increased neuronal discharge rates. Inhibitory interneurons of the hippocampal formation control both neuronal synchronization as well as the global level of activity and are therefore of crucial importance for epilepsy. Recent evidence suggests that changes in synaptic inhibition during temporal lobe epilepsy are rather specific, resulting from selective death or alteration of interneurons in specific hippocampal layers. Hence, epilepsy-induced changes have to be analysed separately for different types of interneurons. Here, we focused on GABAergic neurons located at the border between stratum radiatum and stratum lacunosum-moleculare of hippocampal area CA1 (SRL interneurons), which are included in feedforward inhibitory circuits. In chronically epileptic rats at 6-8 months after pilocarpine-induced status epilepticus, frequencies of spontaneous and miniature inhibitory postsynaptic currents were reduced, yielding an almost three-fold increase in excitation-inhibition ratio. Consistently, action potential frequency of SRL interneurons was about two-fold enhanced. Morphological alterations of the interneurons indicate that these functional changes were accompanied by remodelling of the local network, probably resulting in a loss of functional inhibitory synapses without conceivable cell death. Our data indicate a strong increase in activity of interneurons in dendritic layers of the chronically epileptic CA1 region. This alteration may enhance feedforward inhibition and rhythmogenesis and--together with specific changes in other interneurons--contribute to seizure susceptibility and pathological synchronization.

Allosteric enhancement of metabotropic glutamate receptor 5 function promotes spatial memory.

Group I metabotropic glutamate receptors (mGluRs) have been implicated in learning and memory formation. Recent findings indicate an important function of the group I mGluR subtype 5. Here, we used the Y-maze spatial alternation task and examined whether enhancement of intrinsic mGluR5 activity immediately after learning, i.e. during a critical period for memory consolidation, would have any consequences on long-term memory retention in rats. Intracerebroventricular application of the allosteric mGluR5 potentiator DFB (3,3'-difluorobenzaldazine) resulted in a marked improvement in spatial alternation retention when it was tested 24 h after training. The promnesic effect increased with the difficulty of the task and was apparently due to a substantial enhancement of consolidation. The applied dose of DFB did not cause behavioral changes in the open field, and was devoid of structural side-effects as evaluated by immunohistochemical examination. Our results suggest an important function of post-training mGluR5 activation in some types of hippocampus-dependent spatial learning.

Interleukin-6: a cytokine to forget.

It is known that proinflammatory cytokines such as interleukin-6 (IL-6) are expressed in the central nervous system (CNS) during disease conditions and affect several brain functions including memory and learning. In contrast to these effects observed during pathological conditions, here we describe a physiological function of IL-6 in the "healthy" brain in synaptic plasticity and memory consolidation. During long-term potentiation (LTP) in vitro and in freely moving rats, IL-6 gene expression in the hippocampus was substantially increased. This increase was long lasting, specific to potentiation, and was prevented by inhibition of N-methyl-D-aspartate receptors with (+/-)-2-amino-5-phosphonopentanoic acid (AP-5). Blockade of endogenous IL-6 by application of a neutralizing anti-IL-6 antibody 90 min after tetanus caused a remarkable prolongation of LTP. Consistently, blockade of endogenous IL-6, 90 min after hippocampus-dependent spatial alternation learning resulted in a significant improvement of long-term memory. In view of the suggested role of LTP in memory formation, these data implicate IL-6 in the mechanisms controlling the kinetics and amount of information storage.

Excitatory and inhibitory neurons express c-Fos in barrel-related columns after exploration of a novel environment.

Recent work has shown that behaviorally meaningful sensory information processing is accompanied by the induction of several transcription factors in the barrel cortex of rodents. It is now generally accepted that stimulus-transcription coupling is an important step in the sequence of events leading to long-term plastic changes in neuronal structure and function. Nevertheless, so far few data are available as to what types of neurons are involved in such a genomic response. Here, we determined the morphological and neurochemical identity of neurons in rat barrel cortex showing a c-Fos-immunoreactive nucleus after exploration of an enriched environment. Double stainings of c-Fos and glial fibrillary acidic protein excluded astrocytes as a possible cell type expressing this transcription factor. By morphological phenotyping with intracellular Lucifer Yellow injections, it was found that a large majority were probably excitatory pyramidal cells, but inhibitory interneurons were also found to contain c-Fos-immunoreactive nuclei. By neurochemical phenotyping of GABAergic interneurons with specific antibodies, a significant induction was found, in a layer-dependent manner, for the populations of glutamic acid decarboxylase-, parvalbumin-, calbindin- and vasoactive intestinal polypeptide-immunoreactive neurons but not for calretinin-immunoreactive cells in experimental compared to control columns. From these data we conclude that thalamic afferents effectively drive cortical excitatory as well as inhibitory intracortical circuits. Thus, the adaptations of receptive field properties of cortical neurons after different manipulations of the sensory periphery are likely to be caused by plastic changes in excitatory and inhibitory networks.

Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptor-deficient mice.

The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PAC1) is a G-protein-coupled receptor binding the strongly conserved neuropeptide PACAP with 1000-fold higher affinity than the related peptide vasoactive intestinal peptide. PAC1-mediated signaling has been implicated in neuronal differentiation and synaptic plasticity. To gain further insight into the biological significance of PAC1-mediated signaling in vivo, we generated two different mutant mouse strains, harboring either a complete or a forebrain-specific inactivation of PAC1. Mutants from both strains show a deficit in contextual fear conditioning, a hippocampus-dependent associative learning paradigm. In sharp contrast, amygdala-dependent cued fear conditioning remains intact. Interestingly, no deficits in other hippocampus-dependent tasks modeling declarative learning such as the Morris water maze or the social transmission of food preference are observed. At the cellular level, the deficit in hippocampus-dependent associative learning is accompanied by an impairment of mossy fiber long-term potentiation (LTP). Because the hippocampal expression of PAC1 is restricted to mossy fiber terminals, we conclude that presynaptic PAC1-mediated signaling at the mossy fiber synapse is involved in both LTP and hippocampus-dependent associative learning.

Distribution of transcript and protein isoforms of the synaptic glycoprotein neuroplastin in rat retina.

PURPOSE: To examine the expression and localization of the neuroplastins (np), two synapse-enriched members of the immunoglobulin (Ig) superfamily of cell-adhesion molecules, in the developing and adult retina and optic nerve. METHODS: Expressions of the two isoforms np55 and np65 and carboxyl-terminal splice variants were investigated by immunocytochemistry, Western blot analysis, RT-PCR, and in situ hybridization. RESULTS: Immunoreactivity for both neuroplastins was confined to the two synaptic layers of the retina: the inner (IPL) and outer plexiform layer (OPL). Significant overlap was found in staining at synaptic structures with synaptophysin. A large proportion of immunoreactivity for both isoforms, however, was of perisynaptic origin. In situ hybridization studies were suggestive of a pre- and postsynaptic localization of np65 in the OPL. Transcripts for np55 were already present at birth in the inner retina, but the hybridization signals increased during postnatal development. Np65 transcripts and immunosignals appeared at later developmental ages, concomitant with synapse formation in the OPL. Several C-terminal neuroplastin cDNA clones harbor an insert of 12 bp, coding for four amino acids (DDEP) in the intracellular domain of neuroplastins. Splice isoforms containing the insert exhibited a developmental expression pattern similar to that of np55; however, both neuroplastins could harbor the C-terminal insert. Neuroplastins were also detected in optic nerve homogenates. RT-PCR and blockade of axonal transport by nerve crush confirmed transcript and protein expression in optic nerve tissue. CONCLUSIONS: The findings suggest a role for neuroplastins in cell adhesion in the plexiform layers during histogenesis, as well as in maintenance of connections between specific cellular structures.

The dendritic architecture of prefrontal pyramidal neurons in schizophrenic patients.

Despite a considerable number of investigations revealing the prefrontal cortex (PFC) to be a major site of pathological changes in schizophrenia, the neuronal basis of these alterations is still unknown. We used a 3-D image analysis technique to investigate the dendritic arborization of Golgi-impregnated prefrontal pyramidal neurons in schizophrenic patients and controls. While the apical dendrites were found to be unchanged in schizophrenics, the basilar dendritic systems were markedly reduced in the patient group. A segment analysis showed that the observed alterations were mainly confined to distal dendritic segments. The dendritic changes are likely to be associated with specific dysfunctions of prefrontal circuitry and point to the pathogenetical relevance of pre- and perinatal disturbances of PFC maturation in schizophrenic patients.

Mitochondrial complex I deficiency in the epileptic focus of patients with temporal lobe epilepsy.

Mitochondria are cellular organelles crucial for energy supply and calcium homeostasis in neuronal cells, and their dysfunction causes seizure activity in some rare human epilepsies. To directly test whether mitochondrial respiratory chain enzymes are abnormal in the most common form of chronic epilepsy, temporal lobe epilepsy (TLE), living human brain specimens from 57 epileptic patients and 2 nonepileptic controls were investigated. In TLE patients with a hippocampal epileptic focus, we demonstrated a specific deficiency of complex I of the mitochondrial respiratory chain in the hippocampal CA3 region. In contrast, TLE patients with a parahippocampal epileptic focus showed reduced complex I activity only in parahippocampal tissue. Inhibitor titrations of the maximal respiration rate of intact human brain slices revealed that the observed reduction in complex I activity is sufficient to affect the adenosine triphosphate production rate. The abnormal complex I activity in the hippocampal CA3 region was paralleled by increased succinate dehydrogenase staining of neurons and marked ultrastructural abnormalities of mitochondria. Therefore, mitochondrial dysfunction is suggested to be specific for the epileptic focus and may constitute a pathomechanism contributing to altered excitability and selective neuronal vulnerability in TLE.

Deletion of the ryanodine receptor type 3 (RyR3) impairs forms of synaptic plasticity and spatial learning.

Deletion of the ryanodine receptor type 3 (RyR3) results in specific changes in hippocampal synaptic plasticity, without affecting hippocampal morphology, basal synaptic transmission or presynaptic function. Robust long-term potentiation (LTP) induced by repeated, strong tetanization in the CA1 region and in the dentate gyrus was unaltered in hippocampal slices in vitro, whereas weak forms of plasticity generated by either a single weak tetanization or depotentiation of a robust LTP were impaired. These distinct physiological deficits were paralleled by a reduced flexibility in re-learning a new target in the water-maze. In contrast, learning performance in the acquisition phase and during probe trial did not differ between the mutants and their wild-type littermates. In the open-field, RyR3(-/-) mice displayed a normal exploration and habituation, but had an increased speed of locomotion and a mild tendency to circular running. The observed physiological and behavioral effects implicate RyR3-mediated Ca(2+) release in the intracellular processes underlying spatial learning and hippocampal synaptic plasticity.

Cellular pathology of hilar neurons in Ammon's horn sclerosis.

In addition to functionally affected neuronal signaling pathways, altered axonal, dendritic, and synaptic morphology may contribute to hippocampal hyperexcitability in chronic mesial temporal lobe epilepsies (MTLE). The sclerotic hippocampus in Ammon's horn sclerosis (AHS)-associated MTLE, which shows segmental neuronal cell loss, axonal reorganization, and astrogliosis, would appear particularly susceptible to such changes. To characterize the cellular hippocampal pathology in MTLE, we have analyzed hilar neurons in surgical hippocampus specimens from patients with MTLE. Anatomically well-preserved hippocampal specimens from patients with AHS (n = 44) and from patients with focal temporal lesions (non-AHS; n = 20) were studied using confocal laser scanning microscopy (CFLSM) and electron microscopy (EM). Hippocampal samples from three tumor patients without chronic epilepsies and autopsy samples were used as controls. Using intracellular Lucifer Yellow injection and CFLSM, spiny pyramidal, multipolar, and mossy cells as well as non-spiny multipolar neurons have been identified as major hilar cell types in controls and lesion-associated MTLE specimens. In contrast, none of the hilar neurons from AHS specimens displayed a morphology reminiscent of mossy cells. In AHS, a major portion of the pyramidal and multipolar neurons showed extensive dendritic ramification and periodic nodular swellings of dendritic shafts. EM analysis confirmed the altered cellular morphology, with an accumulation of cytoskeletal filaments and increased numbers of mitochondria as the most prominent findings. To characterize cytoskeletal alterations in hilar neurons further, immunohistochemical reactions for neurofilament proteins (NFP), microtubule-associated proteins, and tau were performed. This analysis specifically identified large and atypical hilar neurons with an accumulation of low weight NFP. Our data demonstrate striking structural alterations in hilar neurons of patients with AHS compared with controls and non-sclerotic MTLE specimens. Such changes may develop during cellular reorganization in the epileptogenic hippocampus and are likely to contribute to the pathogenesis or maintenance of temporal lobe epilepsy.

Proline-rich synapse-associated protein-1/cortactin binding protein 1 (ProSAP1/CortBP1) is a PDZ-domain protein highly enriched in the postsynaptic density.

The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neurotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse formation in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain development. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and immunoelectron microscopy in situ revealed a strong association with PSDs of hippocampal excitatory synapses. The accumulation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal development, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punctate staining is observed. At the ultrastructural level, the immunoreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/CortBP1 may be involved in the assembly of the PSD during neuronal differentiation.

Presynaptic localization of the PACAP-typeI-receptor in hippocampal and cerebellar mossy fibres.

The distribution of PACAP-typeI-receptor (PACAP-I-R) mRNA and protein was studied in mouse using probes and a newly developed antiserum recognizing all known splice variants. RNase protection assays revealed highest expression levels of PACAP-I-R mRNA in brain, in particular the hypothalamus and hippocampus. At the cellular level, in situ hybridization analysis demonstrated widespread distribution of PACAP-I-R mRNA in neurons throughout the brain, while glial cells did not express the gene. Highest expression levels of PACAP-I-R mRNA were observed in three regions: the limbic system, the hypothalamus, and the brainstem. In accordance with data obtained from in situ hybridization analysis, immunohistochemistry showed widespread distribution of PACAP-I-R like immunoreactivity in the neuropil. Rather strong immunoreactivity was found in cerebellar and hippocampal mossy fibres where double immunolabelling revealed the presynaptic localization of the receptor protein. At the ultrastructural level, PACAP-I-R like immunoreactivity was observed around synaptic vesicles and close to the presynaptic grid in hippocampal mossy fibre terminals. This finding is in contradiction to the described postsynaptic localization of the PACAP-I-R in dendritic processes of hippocampal granule cells in rat. Due to their presynaptic induction, mossy fibre LTPs are distinctly different from LTPs in all other hippocampal regions. Therefore, the presynaptic localization of the PACAP-I-R in mossy fibre terminals may implicate this gene in influencing the synaptic strength of the mossy fibre pathway and hence memory consolidation.

Loss of beta1 integrin function results in a retardation of myogenic, but an acceleration of neuronal, differentiation of embryonic stem cells in vitro.

Integrin cell surface receptors play an important role for cell adhesion, migration, and differentiation during embryonic development by mediating cell-cell and cell-matrix interactions. Less is known about the function of integrins during commitment and lineage determination of early embryogenesis. Homozygous inactivation of the beta1 integrin gene results in embryonal death in mice around the time of implantation. In vitro, differentiation of embryonic stem (ES) cells which lack beta1 integrin (beta1-/-) into the cardiogenic lineage is delayed and results in a disordered cellular specification (Fässler et al., J. Cell Sci. 109, 2989-2999, 1996). To analyze beta1 integrin function during myogenesis and neurogenesis we studied differentiation of beta1-/- ES cells via embryoid bodies into skeletal muscle and neuronal cells in vitro. beta1-/- cells showed delayed and reduced myogenic differentiation compared to wildtype and heterozygous (beta1+/-) ES cells. RT-PCR analysis demonstrated delayed expression of skeletal muscle-specific genes in the absence of beta1 integrin. Immunofluorescence studies with antibodies against the sarcomeric proteins myosin heavy chain, titin, nebulin, and slow C-protein showed that myotubes formed, but their number was reduced and the assembly of sarcomeric structures was retarded. In contrast, neuronal cells differentiating from beta1-/- ES cells appeared earlier than wildtype and heterozygous (beta1+/-) ES cells. This was shown by the accelerated expression of neuron-specific genes and an increased number of neuronal cells in beta1-/- embryoid bodies. However, neuronal outgrowth was retarded in the absence of beta1 integrin. No functional difference between wildtype and beta1-/- cells was found with respect to secretion of gamma-aminobutyric acid, the main neurotransmitter of ES cell-derived neuronal cells. The lineage-specific effects of loss of beta1 integrin function, that is the inhibition of mesodermal and acceleration of neuroectodermal differentiation, were supported by differential expression of genes encoding lineage-specific transcription factors (Brachyury, Pax-6, Mash1) and signaling molecules (BMP-4 and Wnt-1). Because of the reduced and delayed expression of the BMP-4 encoding gene in beta1-/- cells, we analyzed in wildtype and beta1-/- cells the regulatory role of exogenously added BMP-4 on the expression of the mesodermal and neuronal marker genes, Brachyury and wnt-1, respectively. The data suggest that BMP-4 plays a regulatory role during differentiation of wildtype and beta1-/- cells by modifying mesodermal and neuronal pathways. The reduced expression of BMP-4 in beta1-/- cells may account for the accelerated neuronal differentiation in beta1-/- ES cells.

Sustained long term potentiation and anxiety in mice lacking the Mas protooncogene.

The Mas protooncogene is a maternally imprinted gene encoding an orphan G protein-coupled receptor expressed mainly in forebrain and testis. Here, we provide evidence for a function of Mas in the central nervous system. Targeted disruption of the Mas protooncogene leads to an increased durability of long term potentiation in the dentate gyrus, without affecting hippocampal morphology, basal synaptic transmission, and presynaptic function. In addition, Mas-/- mice show alterations in the onset of depotentiation. The permissive influence of Mas ablation on hippocampal synaptic plasticity is paralleled by behavioral changes. While spatial learning in the Morris water maze is not significantly influenced, Mas-deficient animals display an increased anxiety as assessed in the elevated-plus maze. Thus, Mas is an important modulating factor in the electrophysiology of the hippocampus and is involved in behavioral pathways in the adult brain.

Formation of postsynaptic-like membranes during differentiation of embryonic stem cells in vitro.

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, alpha 1 subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the gamma- and epsilon-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR epsilon-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiation in vitro neuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by "loss of function" analysis in vitro.

Some functions of primary auditory cortex in learning and memory formation.

In the primary auditory field AI of gerbil auditory cortex, aversive tone conditioning paradigms reshaped frequency receptive fields of single units and also changed the spatial representation of tones in fluoro-2-deoxyglucose (FDG) experiments. As another aspect of learning-induced plasticity in gerbil AI, antibodies against the immediate early gene product c-Fos identified an unusual spatial pattern of neurons in terms of a "macrocolumn." The pattern resulted from repeated short exposure of the animals to a tone in a new environment. The search for transmitters that may mediate this gene activation is carried out by microdialysis through chronically implanted probes in auditory cortex. So far, dopamine transmission was found to reflect specific aspects of auditory learning in cortex. The results suggest that spectral features of sounds as well as aspects of learned behavioral meaning of the sounds may be represented in AI.