5-HT7 receptors modulate GABAergic transmission in rat hippocampal CA1 area

The effects of the activation of serotonin-7 (5-HT(7)) receptors were investigated in the CA1 area pyramidal cells and stratum radiatum fast spiking GABAergic interneurons of rat hippocampal slices. To activate 5-HT(7) receptors, 5-carboxamidotryptamine (5-CT), a nonselective 5-HT(1A)/5-HT(7) agonist, was applied in the presence of N-[2-[4-(2-methoxyphenyl)-1piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY 100635), a selective 5-HT(1A) receptor antagonist. The activation of 5-HT(7) receptors resulted in a dose-dependent increase in the mean frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from pyramidal neurons while the mean amplitude of sIPSCs remained unaltered. A nonselective glutamate receptor antagonist, kynurenic acid, and voltage-gated sodium channel blocker, tetrodotoxin (TTX), attenuated but did not prevent the 5-HT(7) receptor-mediated increase of sIPSCs frequency in pyramidal cells. 5-CT application did not influence the excitability of stratum radiatum interneurons but it dose-dependently increased the mean frequency of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from interneurons while the mean amplitude of sEPSCs remained unaltered. These data suggest that the activation of 5-HT(7) receptors results in an enhancement of the GABAergic transmission in the hippocampal CA1 area via two mechanisms. The first one involves an enhancement of excitatory glutamatergic input to GABAergic interneurons and is likely to be mediated by presynaptic 5-HT(7) receptors. The second effect, most likely related to the activation of 5-HT(7) receptors located on interneurons, results in an enhancement of the release of GABA.     

Effects of methylprednisolone on the GABA- and glutamate-induced currents: Relevance to glucocorticoid-induced neurotoxicity and brain aging

We have previously shown that both epidural administration and microinjection of methylprednisolone (MP) produces neuronal hyperexcitability in the murine spinal cord in vivo. In this study, the whole-cell patch-clamp technique was used to describe and characterize MP-induced neuronal hyperexcitability. Exposure of 10- to 18-day old dissociated spinal cord cultures to 65 μM-8 mM MP caused a concentration-dependent increase in the firing rate. MP (1 mM) increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs). The amplitude of the sEPSCs was also increased in response to 1 mM MP, whereas sIPSCs became smaller in size in the presence of MP. MP (1 mM) reduced the amplitude of the γ-aminobutyric acid (GABA)-induced currents, whereas it increased the amplitude of the glutamate-induced currents. And finally, MP (1 mM), by itself, did not change the overall postsynaptic membrane conductance. These observations suggest that (1) MP can act as an excitatory agent in vitro, (2) it can act at the presynaptic as well as the postsynaptic level, and (3) it affects spinal cord neurons by influencing the ligand-gated (GABA and glutamate) channels.     

Effects of Tramadol on Substantia Gelatinosa Neurons in the Rat Spinal Cord: An In Vivo Patch-Clamp Analysis

Tramadol is thought to modulate synaptic transmissions in the spinal dorsal horn mainly by activating µ-opioid receptors and by inhibiting the reuptake of monoamines in the CNS. However, the precise mode of modulation remains unclear. We used an in vivo patch clamp technique in urethane-anesthetized rats to determine the antinociceptive mechanism of tramadol. In vivo whole-cell recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) were made from substantia gelatinosa (SG) neurons (lamina II) at holding potentials of 0 mV and -70 mV, respectively. The effects of intravenous administration (0.5, 5, 15 mg/kg) of tramadol were evaluated. The effects of superfusion of tramadol on the surface of the spinal cord and of a tramadol metabolite (M1) were further analyzed. Intravenous administration of tramadol at doses >5 mg/kg decreased the sEPSCs and increased the sIPSCs in SG neurons. These effects were not observed following naloxone pretreatment. Tramadol superfusion at a clinically relevant concentration (10 µM) had no effect, but when administered at a very high concentration (100 µM), tramadol decreased sEPSCs, produced outward currents, and enhanced sIPSCs. The effects of M1 (1, 5 mg/kg intravenously) on sEPSCs and sIPSCs were similar to those of tramadol at a corresponding dose (5, 15 mg/kg). The present study demonstrated that systemically administered tramadol indirectly inhibited glutamatergic transmission, and enhanced GABAergic and glycinergic transmissions in SG neurons. These effects were mediated primarily by the activation of μ-opioid receptors. M1 may play a key role in the antinociceptive mechanisms of tramadol.     

The Reduction of EPSC Amplitude in CA1 Pyramidal Neurons by the Peroxynitrite Donor SIN-1 Requires Ca2+ Influx Via Postsynaptic Non-L-Type Voltage Gated Calcium Channels

The peroxynitrite free radical (ONOO^?) modulation of miniature excitatory postsynaptic currents (mEPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) was investigated in rat CA1 pyramidal neurons using the whole-cell patch clamp technique. SIN-1(3-morpholino-sydnonimine), which can lead the simultaneous generation of superoxide anion and nitric oxide, and then form the highly reactive species ONOO^?, induced dose-dependent inhibition in amplitudes of both mEPSCs and sEPSCs. The SIN-1 action on mEPSC amplitude was completely blocked by U0126, a selective MEK inhibitor, suggesting that MEK contributed to the action of ONOO^? on mEPSCs. The effect of SIN-1 was completely occluded either in the presence of the calcium chelator EGTA or the non-selective calcium channel antagonist Cd²⁺. Furthermore, the application of nifedipine (20 μM), the L-type calcium channel blocker, had no effect on the ONOO^?-induced decrease in mEPSC amplitude, excluding a role for L-type voltage-gated Ca²⁺ channels in this process. SIN-1 inhibited the frequency of sEPSCs but had no effect on mEPSC frequency, which suggested a presynaptic action potential-dependent the action of ONOO^? at CA1 pyramidal neuron synapses. The best-known glutamatergic input to CA1 pyramidal neurons is via Schaffer collaterals from CA3 area. However, no changes were observed in slices treated with SIN-1 on the spontaneous firing rates of CA3 pyramidal neurons. These findings suggested that SIN-1 inhibited glutamatergic synaptic transmission of CA1 pyramidal neurons by a postsynaptic non-L-type voltage gated calcium channel-dependent mechanism.     

Neuronal MHC Class I Molecules are Involved in Excitatory Synaptic Transmission at the Hippocampal Mossy Fiber Synapses of Marmoset Monkeys

Several recent studies suggested a role for neuronal major histocompatibility complex class I (MHCI) molecules in certain forms of synaptic plasticity in the hippocampus of rodents. Here, we report for the first time on the expression pattern and functional properties of MHCI molecules in the hippocampus of a nonhuman primate, the common marmoset monkey (Callithrix jacchus). We detected a presynaptic, mossy fiber-specific localization of MHCI proteins within the marmoset hippocampus. MHCI molecules were present in the large, VGlut1-positive, mossy fiber terminals, which provide input to CA3 pyramidal neurons. Furthermore, whole-cell recordings of CA3 pyramidal neurons in acute hippocampal slices of the common marmoset demonstrated that application of antibodies which specifically block MHCI proteins caused a significant decrease in the frequency, and a transient increase in the amplitude, of spontaneous excitatory postsynaptic currents (sEPSCs) in CA3 pyramidal neurons. These findings add to previous studies on neuronal MHCI molecules by describing their expression and localization in the primate hippocampus and by implicating them in plasticity-related processes at the mossy fiber–CA3 synapses. In addition, our results suggest significant interspecies differences in the localization of neuronal MHCI molecules in the hippocampus of mice and marmosets, as well as in their potential function in these species.     

Dynamic control of glutamatergic synaptic input in the spinal cord by muscarinic receptor subtypes defined using knockout mice

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M(2), M(3), and M(4)) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ～50% neurons from M(3)-single KO and M(1)/M(3) double-KO mice. In addition, the M(3) antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M(5)-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M(2)/M(4) double-KO mice, but not M(2)- or M(4)-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M(2)/M(4) antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M(2) and M(4) receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M(5) is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord.     

Effects of Etomidate on GABAergic and Glutamatergic Transmission in Rat Thalamocortical Slices

Although accumulative evidence indicates that the thalamocortical system is an important target for general anesthetics, the underlying mechanisms of anesthetic action on thalamocortical neurotransmission are not fully understood. The aim of the study is to explore the action of etomidate on glutamatergic and GABAergic transmission in rat thalamocortical slices by using whole cell patch-clamp recording. We found that etomidate mainly prolonged the decay time of spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs), without changing the frequency. Furthermore, etomidate not only prolonged the decay time of miniature inhibitory postsynaptic currents (mIPSCs) but also increased the amplitude. On the other hand, etomidate significantly decreased the frequency of spontaneous glutamatergic excitatory postsynaptic currents (sEPSCs), without altering the amplitude or decay time in the absence of bicuculline. When GABA_A receptors were blocked using bicuculline, the effects of etomidate on sEPSCs were mostly eliminated. These results suggest that etomidate enhances GABAergic transmission mainly through postsynaptic mechanism in thalamocortical neuronal network. Etomidate attenuates glutamatergic transmission predominantly through presynaptic action and requires presynaptic GABA_A receptors involvement.     

Slower spontaneous excitatory postsynaptic currents in spiny versus aspiny hilar neurons.

In the hilar region of the rat hippocampus, large spontaneous excitatory postsynaptic currents (sEPSCs) mediated by non-NMDA glutamate receptors are present in both excitatory spiny mossy cells and inhibitory aspiny hilar interneurons, making these neurons ideal candidates for a comparative study using the tight seal whole-cell recording technique. Although sEPSCs have similar amplitude distributions, the rise and decay times are significantly slower in spiny versus aspiny neurons. Similar kinetic differences are observed in synaptic currents evoked by mossy fiber stimulation. These results demonstrate a physiological difference between the excitatory drive to excitatory and inhibitory neurons in the hilus that certainly contributes to differences in synaptic strength and that may be applicable to other brain regions. Furthermore, since the development or modification of individual spines or groups of spines may affect synaptic strength, these results may be pivotal in establishing a role for spines in modulating synaptic activity.     

Glucose Rapidly Induces Different Forms of Excitatory Synaptic Plasticity in Hypothalamic POMC Neurons

Hypothalamic POMC neurons are required for glucose and energy homeostasis. POMC neurons have a wide synaptic connection with neurons both within and outside the hypothalamus, and their activity is controlled by a balance between excitatory and inhibitory synaptic inputs. Brain glucose-sensing plays an essential role in the maintenance of normal body weight and metabolism; however, the effect of glucose on synaptic transmission in POMC neurons is largely unknown. Here we identified three types of POMC neurons (EPSC(+), EPSC(−), and EPSC(+/−)) based on their glucose-regulated spontaneous excitatory postsynaptic currents (sEPSCs), using whole-cell patch-clamp recordings. Lowering extracellular glucose decreased the frequency of sEPSCs in EPSC(+) neurons, but increased it in EPSC(−) neurons. Unlike EPSC(+) and EPSC(−) neurons, EPSC(+/−) neurons displayed a bi-phasic sEPSC response to glucoprivation. In the first phase of glucoprivation, both the frequency and the amplitude of sEPSCs decreased, whereas in the second phase, they increased progressively to the levels above the baseline values. Accordingly, lowering glucose exerted a bi-phasic effect on spontaneous action potentials in EPSC(+/−) neurons. Glucoprivation decreased firing rates in the first phase, but increased them in the second phase. These data indicate that glucose induces distinct excitatory synaptic plasticity in different subpopulations of POMC neurons. This synaptic remodeling is likely to regulate the sensitivity of the melanocortin system to neuronal and hormonal signals.     

Expression of nerve growth factor and its receptors in the somatosensory-motor cortex of Macaca nemestrina

The present study was designed to examine the nerve growth factor (NGF) system (ligand and receptor-expressing neurons) in the somatosensory (areas 1, 3a, and 3b) and motor (area 4) cortices of the mature macaque. Light and electron microscope immunohistochemistry was used to assess the distribution and identity of NGF-, p75-, and trk-expressing elements. In each cortical area examined, NGF-positive neuronal somata were distributed through all laminae; most immunolabeled neurons were in layers II, III, and V. Based upon light microscope criteria (e.g., the morphology of proximal dendrites), both pyramidal and stellate neurons expressed NGF. Of the identifiable NGF- immunoreactive cells, 92% were pyramidal neurons and the remainder was stellate neurons. The electron microscope study showed that most (88%) NGF-positive somata formed symmetric synapses, whereas the others formed both symmetric and asymmetric synapses. As the somata of pyramidal neurons form only symmetric synapses and those of inhibitory stellate neurons form both symmetric and asymmetric somatic synapses, the ultrastructural data support the light microscopic analyses. In contrast, neurotrophin receptors, p75 and trk, were expressed chiefly by the cell bodies of layer V pyramidal neurons and the supragranular neuropil. At the ultrastructural level, receptor-positive profiles were post-synaptic elements (e.g., dendritic shafts and spines) and the concentration of immunoreactivity was greatest in the vicinity of post-synaptic densities. Thus, NGF regulatory systems parallel excitatory and inhibitory neurotransmitter systems. Cortex contains the morphological framework by which pyramidal and/or inhibitory stellate neurons can affect the activity of post-synaptic pyramidal neurons via anterograde and autocrine/paracrine NGF systems.     

Neogenesis of cerebellar Purkinje neurons from gene-marked bone marrow cells in vivo.

The versatility of stem cells has only recently been fully recognized. There is evidence that upon adoptive bone marrow (BM) transplantation (BMT), donor-derived cells can give rise to neuronal phenotypes in the brains of recipient mice. Yet only few cells with the characteristic shape of neurons were detected 1-6 mo post-BMT using transgenic or newborn mutant mice. To evaluate the potential of BM to generate mature neurons in adult C57BL/6 mice, we transferred the enhanced green fluorescent protein (GFP) gene into BM cells using a murine stem cell virus-based retroviral vector. Stable and high level long-term GFP expression was observed in mice transplanted with the transduced BM. Engraftment of GFP-expressing cells in the brain was monitored by intravital microscopy. In a long-term follow up of 15 mo post-BMT, fully developed Purkinje neurons were found to express GFP in both cerebellar hemispheres and in all chimeric mice. GFP-positive Purkinje cells were also detected in BM chimeras from transgenic mice that ubiquitously express GFP. Based on morphologic criteria and the expression of glutamic acid decarboxylase, the newly generated Purkinje cells were functional.     

Focal Cortical Lesions Induce Bidirectional Changes in the Excitability of Fast Spiking and Non Fast Spiking Cortical Interneurons

A physiological brain function requires neuronal networks to operate within a well-defined range of activity. Indeed, alterations in neuronal excitability have been associated with several pathological conditions, ranging from epilepsy to neuropsychiatric disorders. Changes in inhibitory transmission are known to play a key role in the development of hyperexcitability. However it is largely unknown whether specific interneuronal subpopulations contribute differentially to such pathological condition. In the present study we investigated functional alterations of inhibitory interneurons embedded in a hyperexcitable cortical circuit at the border of chronically induced focal lesions in mouse visual cortex. Interestingly, we found opposite alterations in the excitability of non fast-spiking (Non Fs) and fast-spiking (Fs) interneurons in acute cortical slices from injured animals. Non Fs interneurons displayed a depolarized membrane potential and a higher frequency of spontaneous excitatory postsynaptic currents (sEPSCs). In contrast, Fs interneurons showed a reduced sEPSCs amplitude. The observed downscaling of excitatory synapses targeting Fs interneurons may prevent the recruitment of this specific population of interneurons to the hyperexcitable network. This mechanism is likely to seriously affect neuronal network function and to exacerbate hyperexcitability but it may be important to protect this particular vulnerable population of GABAegic neurons from excitotoxicity.     

The functional upregulation of piriform cortex is associated with cross-modal plasticity in loss of whisker tactile inputs

Background

Cross-modal plasticity is characterized as the hypersensitivity of remaining modalities after a sensory function is lost in rodents, which ensures their awareness to environmental changes. Cellular and molecular mechanisms underlying cross-modal sensory plasticity remain unclear. We aim to study the role of different types of neurons in cross-modal plasticity.

Methodology/Principal Findings

In addition to behavioral tasks in mice, whole-cell recordings at the excitatory and inhibitory neurons, and their two-photon imaging, were conducted in piriform cortex. We produced a mouse model of cross-modal sensory plasticity that olfactory function was upregulated by trimming whiskers to deprive their sensory inputs. In the meantime of olfactory hypersensitivity, pyramidal neurons and excitatory synapses were functionally upregulated, as well as GABAergic cells and inhibitory synapses were downregulated in piriform cortex from the mice of cross-modal sensory plasticity, compared with controls. A crosswire connection between barrel cortex and piriform cortex was established in cross-modal plasticity.

Conclusion/Significance

An upregulation of pyramidal neurons and a downregulation of GABAergic neurons strengthen the activities of neuronal networks in piriform cortex, which may be responsible for olfactory hypersensitivity after a loss of whisker tactile input. This finding provides the clues for developing therapeutic strategies to promote sensory recovery and substitution.     

A computer model of generation of the motor cortex neuron processes seen in the course of execution of instrumental movement

A computer model of neuronal processes in the motor cortex column is presented. The model is consisted of two pyramidal cell layers with two groups of inhibitory interneurons, selectively controlling pyramidal cell soma and dendrite, in each. Active Na, Ca and K conductances are included in the model of a single neuron. Horizontal excitatory connections between pyramidal cells in the upper layer are largely of NMDA-receptor type, that in the lower layer--of non-NMDA-type. All inhibitory synapses are of GABA(A)-type. The model reproduces the main phenomenon observed in the motor cortex during the execution of conditioned movements. Consequent to an early excitation the upper layer pyramidal cells generate a late NMDA-dependent reflexive response to afferent conditional stimulation, which as in a real case is diminished by GABA(A)-type synaptic inhibition and afferent stimulus strength increase. The characteristic inverse relation between the late response manifestation and the stimulus strength observed in the real cortex can be reproduced in the model only if NMDA-glutamate receptors were preferentially localized in the terminals of pyramidal cell backward collaterals, not in the terminals of the afferent fibers on pyramidal neurons. The intended component of motor cortex neuronal activity is generated in NMDA-independent manner by the pyramidal cells of lower layer. The slow time coarse of intended component as compared with short duration of AMPA epsp's is due to a consecutive relay-race--like activation of pyramidal neurons with different dendrit-to-soma ratio.     

Aberrant GABA(A) receptor-mediated inhibition in cortico-thalamic networks of succinic semialdehyde dehydrogenase deficient mice

Aberrant γ-aminobutyric acid type A (GABA(A)) receptor-mediated inhibition in cortico-thalamic networks remains an attractive mechanism for typical absence seizure genesis. Using the whole-cell patch clamp technique we examined 'phasic' and 'tonic' GABA(A) inhibition in thalamocortical neurons of somatosensory (ventrobasal, VB) thalamus, nucleus reticularis thalami (NRT) neurons, and layer 5/6 pyramidal neurons of the somatosensory (barrel) cortex of succinic semialdehyde dehydrogenase (SSADH) knock-out (SSADH(-/-)) mice that replicate human SSADH deficiency and exhibit typical absence seizures. We found increased sIPSC frequency in both VB and NRT neurons and larger sIPSC amplitude in VB neurons of SSADH(-/-) mice compared to wild-type animals, demonstrating an increase in total phasic inhibition in thalamus of SSADH(-/-) mice. mIPSCs in both VB and NRT neurons were no different between genotypes, although there remained a trend toward more events in SSADH(-/-) mice. In cortical layer 5/6 pyramidal neurons, sIPSCs were fewer but larger in SSADH(-/-) mice, a feature retained by mIPSCs. Tonic currents were larger in both thalamocortical neurons and layer 5/6 pyramidal neurons from SSADH(-/-) mice compared to WTs. These data show that enhanced, rather than compromised, GABA(A) receptor-mediated inhibition occurs in cortico-thalamic networks of SSADH(-/-) mice. In agreement with previous studies, GABA(A) receptor-mediated inhibitory gain-of-function may be a common feature in models of typical absence seizures, and could be of pathological importance in patients with SSADH deficiency.     

Identification of 5-HT receptor subtypes enhancing inhibitory transmission in the rat spinal dorsal horn in vitro

ABSTRACT: BACKGROUND: 5-hydroxytryptamine (5-HT) is one of the major neurotransmitters widely distributed in the CNS. Several 5-HT receptor subtypes have been identified in the spinal dorsal horn which act on both pre- and postsynaptic sites of excitatory and inhibitory neurons. However, the receptor subtypes and sites of actions as well as underlying mechanism are not clarified rigorously. Several electrophysiological studies have been performed to investigate the effects of 5-HT on excitatory transmission in substantia gelatinosa (SG) of the spinal cord. In the present study, to understand the effects of 5-HT on the inhibitory synaptic transmission and to identify receptor subtypes, the blind whole cell recordings were performed from SG neurons of rat spinal cord slices. RESULTS: Bath applied 5-HT (50 microM) increased the frequency but not amplitudes of spontaneous inhibitory postsynaptic currents (sIPSCs) in 58% of neurons, and both amplitude and frequency in 23 % of neurons. The frequencies of GABAergic and glycinergic mIPSCs were both enhanced. TTX (0.5 microM) had no effect on the increasing frequency, while the enhancement of amplitude of IPSCs was eliminated. Evoked-IPSCs (eIPSCs) induced by focal stimulation near the recording neurons in the presence of CNQX and APV were enhanced in both amplitude by 5-HT. In the presence of Ba2+ (1 mM), a potassium channel blocker, 5-HT had no effect on both frequency and amplitude. A 5-HT2Areceptor agonist, TCB-2 mimicked the 5-HT effect, and ketanserin, an antagonist of 5-HT2A receptor, inhibited the effect of 5-HT partially and TCB-2 almost completely. A 5-HT2C receptor agonist WAY 161503 mimicked the 5-HT effect and this effect was blocked by a 5-HT2C receptor antagonist, N-desmethylclozapine. The amplitude of sIPSCs were unaffected by both agonists. A 5-HT3 receptor agonist mCPBG enhanced both amplitude and frequency of sIPSCs. This effect was blocked by a 5-HT3 receptor antagonist ICS-205,930. The perfusion of 5-HT2B receptor agonist had no effect on sIPSCs. CONCLUSIONS: Our results demonstrated that 5-HT modulated the inhibitory transmission in SG by the activation of 5-HT2A and 5-HT2C receptors subtypes located predominantly at inhibitory interneuron terminals, and 5-HT3 receptors located at inhibitory interneuron terminals and soma-dendrites, consequently enhanced both frequency and amplitude.     

Genetic selection of sox1GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation

Because of their ability to proliferate and to differentiate into diverse cell types, embryonic stem (ES) cells are a potential source of cells for transplantation therapy of various diseases, including Parkinson's disease. A critical issue for this potential therapy is the elimination of undifferentiated cells that, even in low numbers, could result in teratoma formation in the host brain. We hypothesize that an efficient solution would consist of purifying the desired cell types, such as neural precursors, prior to transplantation. To test this hypothesis, we differentiated sox1-green fluorescent protein (GFP) knock-in ES cells in vitro, purified neural precursor cells by fluorescence-activated cell sorting (FACS), and characterized the purified cells in vitro as well as in vivo. Immunocytofluorescence and RT-PCR analyses showed that this genetic purification procedure efficiently removed undifferentiated pluripotent stem cells. Furthermore, when differentiated into mature neurons in vitro, the purified GFP+ cell population generated enriched neuronal populations, whereas the GFP- population generated much fewer neurons. When treated with dopaminergic inducing signals such as sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF8), FACS-purified neural precursor cells responded to these molecules and generated dopaminergic neurons as well as other neural subtypes. When transplanted, the GFP+ cell population generated well contained grafts containing dopaminergic neurons, whereas the GFP- population generated significantly larger grafts (about 20-fold) and frequent tumor-related deaths in the transplanted animals. Taken together, our results demonstrate that genetic purification of neural precursor cells using FACS isolation can effectively remove unwanted proliferating cell types and avoid tumor formation after transplantation.     

Long-latency responses to conditioned stimulus in the cat motor cortex can be generated through the enhancement of efficiency excitatory collaterals of pyramidal neuron

The long-latency excitatory components are the characteristic feature of neuronal responses to conditional stimuli in the motor cortex of the cat. The data presented suggest that the neuronal machine that generates these reactions is that, generating long-latency epileptiform discharges in epileptogenic cortex. The long-latency component generation is based on NMDA-receptor activation in the recurrent excitatory collaterals of the cortical pyramidal neurons. The response delay is dependent on initial activation of inhibitory GABA(A) receptors. The emergence of the late components in the course of motor learning take place as a result of efficiency enhancement of recurrent collaterals synaptic linkage with pyramidal neurons.     

Is the Outgrowth of Transplant-Derived Axons Guided by Host Astrocytes and Myelin Loss?

Loss of cortical neurons may lead to sever and sometimes irreversible deficits in motor function in a number of neuropathological conditions. Absence of spontaneous axonal regeneration following trauma in the adult central nervous system (CNS) is attributed to inhibitory factors associated to the CNS white matter and to the non-permissive environment provided by reactive astrocytes that form a physical and biochemical barrier scar. Neural transplantation of embryonic neurons has been widely assessed as a potential approach to overcome the generally limited capacity of the mature CNS to regenerate axons or to generate new neurons in response to cell loss. We have recently shown that embryonic (E14) mouse motor cortical tissue transplanted into the damaged motor cortex of adult mice developed efferent projections to appropriate cortical and subcortical host targets including distant areas such as the spinal cord, with a topographical organization similar to that of intact motor cortex. Several parameters might account for the outgrowth of axonal projections from embryonic neurons within a presumably non-permissive adult brain, among which are astroglial reactions and myelin formation. In the present study, we have examined the role of astrocytes and myelin in the axonal outgrowth of transplanted neurons.Key Words: motor cortex, neuronal transplantation, embryonic cells, GFP, GFAP, PLP