Excitability changes in lumbosacral spinothalamic neurons produced by non-noxious mechanical stimuli and by graded electrical stimuli applied to the face

The changes in the excitability of lumbosacral spinothalamic neurons produced by activating afferents in the trigeminal nerve using electrical or mechanical stimuli was investigated in cats anesthetized with alpha-chloralose. In most spinothalamic neurons, weak electrical stimuli or step indentations of the skin of the face produced an increase followed by decrease in the excitability of these cells. In experiments in which the effect of activating specific groups of trigeminal afferent fibers on these excitability changes was evaluated, the suppression could be produced by activating only the fastest conducting cutaneous afferent fibers. Step indentations of the facial skin affected the excitability of spinothalamic neurons in a manner similar to electrical stimuli. The duration of the suppression phase appeared to be largely independent of the duration of the step indentation of the facial skin. It was concluded that the descending system mediating the suppression phase is activated largely by cutaneous afferents from rapidly adapting receptors. The effects of subtotal spinal cord lesions on the excitation and suppression phases produced by facial stimulation indicate that the pathways mediating the supppression descend bilaterally in the dorsal part of the lateral fasciculus. The excitation phase appears to be mediated largely by pathways in the dorsal part of the ipsilateral lateral fasciculus.     

Modulation of terminal excitability of mesolimbic dopaminergic neurons by D-amphetamine and haloperidol

Electrophysiological techniques were used to study the changes in the terminal excitability of mesolimbic DA and non-DA neurons following the infusion of D-amphetamine (D-AMP) and haloperidol (HAL) into the nucleus accumbens (NAc) of rats. The amount of current needed to evoke antidromic spikes by electrical stimulation of the NAc was used as an index of the excitability of axon terminals of these neurons. The excitability of DA neurons was decreased by D-AMP and increased by HAL. In addition, the effect produced by D-AMP was reversed by HAL. By contrast, these drugs either induced an opposite effect or were ineffective in inducing changes on the excitability of nerve terminals of mesolimbic non-DA neurons. Infusion of the vehicle or saline produced no effect. D-AMP and HAL were still effective in modulating the excitability of mesolimbic DA nerve terminals after the destruction of NAc neurons by ibotenic acid. The results suggest that the effects seen after D-AMP and HAL are mediated primarily by DA autoreceptors. It is likely that the increase in the current needed for evoking antidromic spikes after infusion of D-AMP into the terminal region is the consequence of DA autoreceptor-mediated hyperpolarization of terminal membranes. On the other hand, HAL could exert its actions by blocking autoreceptor-mediated hyperpolarization.     

Targeted gene transfer to nigrostriatal neurons in the rat brain by helper virus-free HSV-1 vector particles that contain either a chimeric HSV-1 glycoprotein C-GDNF or a gC-BDNF protein

Direct gene transfer into neurons has potential for both studying neuronal physiology and for developing gene therapy treatments for specific neurological conditions. Due to the heterogeneous cellular composition of the brain, cell-type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. The two prevalent approaches for achieving cell-type-specific expression are to use a cell-type-specific promoter to control recombinant gene expression or to modify a virus vector particle to target gene transfer to a specific cell type. Targeted gene transfer to multiple peripheral cell types has been described, but targeted gene transfer to a specific type of neuron in the brain has yet to be reported. Targeted gene transfer approaches with Herpes Simplex Virus (HSV-1) vectors have focused on modifying glycoprotein C (gC) to remove the heparin binding domain and add a binding activity for a specific protein on the cell surface. This study was designed to develop HSV-1 vectors that target gene transfer to cells that contain receptors for either glial-cell-line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF), such as nigrostriatal neurons. We isolated chimeric gC-GDNF or chimeric gC-BDNF constructs, and the resulting proteins were incorporated into HSV-1 virus particles. We performed helper virus-free HSV-1 vector packaging in the presence of each chimeric protein. The resulting vector stocks supported 2.2- to 5.0-fold targeted gene transfer to nigrostriatal neurons in the rat brain, compared to vector particles that contained wild-type (wt) gC. Gene transfer to nigrostriatal neurons by vector particles that contained chimeric gC-BDNF was reduced by preincubation with an anti-BDNF antibody. Targeted gene transfer to neurons that contain specific neurotrophic factor receptors may benefit specific physiological and gene therapy studies.     

Development- and experience-dependent plasticity in the dorsomedial habenula

Of the two major subdivisions of the habenula, the medial and lateral nuclei, the medial habenula is the least understood in terms of synaptic transmission, intrinsic properties and plasticity. The medial habenula (MHb) is composed of glutamatergic neurons which receive the majority of their inputs from the septal region and project predominantly to the interpeduncular nucleus (IPN). To understand the synaptic transmission, we studied both glutamatergic and GABAergic synaptic transmission in the dorsal region of the medial habenula (dMHb). While glutamatergic transmission dominates during early development, an attenuation of glutamatergic transmission and an enhancement of GABAergic transmission occur during development leading into adulthood. Furthermore, as reported previously, GABA_A receptor-mediated transmission is excitatory in the adult dMHb, which is consistent with the reduced expression of the K-Cl co-transporter KCC2. Given the potential role of the dMHb in aversive behaviors, we examined whether fear conditioning or exposure to foot shock affects excitability in dMHb neurons. We observed a suppression of the excitability of dMHb neurons in mice that either underwent fear conditioning or were exposed to foot shock. Furthermore, we observed a suppression of GABAergic but not glutamatergic transmission in the dMHb neurons following fear conditioning. These results suggest that aversive experience produces a suppression of the dMHb neuronal activity. Given that the medial habenula is upstream of the median raphe nucleus which is believed to be involved in the negative regulation of aversive memory, the suppression of dMHb neurons following an aversive experience might play a role in strengthening of aversive memories.     

Polyethylenimine-mediated NGF gene delivery protects transected septal cholinergic neurons

Polyethylenimine (PEI) is an effective vehicle for in vivo gene delivery in many tissues including brain. PEI mediates transgene expression in brain neurons and glia. To investigate whether PEI-mediated nerve growth factor (NGF) gene transfer protected axotomized septal cholinergic neurons, we injected linear PEI (in vivo jetPEI, Qbiogene) complexed with a plasmid encoding for mouse NGF (PEI/pNGF-W) into the rat septum. PEI complexed with a plasmid encoding for green fluorescent protein (PEI/pGFP) was used as the control. PEI-mediated gene expression was predominantly neuronal. Fimbria-fornix transections (FFTs), conducted 1 day after rats were injected with control vector, resulted in a 70% loss of septal cholinergic neurons. In contrast, PEI/pNGF-W injection prior to FFTs attenuated the loss of septal cholinergic neurons. This is the first study, to our knowledge, that shows the neuroprotective effects induced by PEI-mediated trophic factor gene transfer in brain.     

Rapid and efficient electroporation-based gene transfer into primary dissociated neurons

Non-viral gene transfer into neurons has proved to be a formidable task. Here, we describe an electroporation-based method that allows efficient and reliable DNA transfer into dissociated neural cells before they are plated and cultured. In hippocampal neural cells derived from either neonatal mouse or embryonic chicken brains, a high transfection rate was already observed 5 h after transfection, and reached 40-80% in 24 h, as monitored by expression of enhanced green fluorescent protein (eGFP). The level of eGFP expression per cell depended on the amount of DNA used in a gene transfer experiment. The survival and neuritic length of transfected cells resembled that of non-electroporated cells. The transfected neurons showed normal immunostaining for endogenous synaptic protein synaptophysin and the neural cell adhesion molecule (NCAM). Furthermore, efficient gene transfer of the NCAM isoform NCAM140 and eGFP-tagged NCAM140 could be achieved, allowing visualization of NCAM140 expression. Also, a glycosylphosphatidylinositol-anchored eGFP could be efficiently expressed, highlighting lipid rafts without altering electrophysiological properties of transfected neurons. When neurons transfected with green and red fluorescent proteins were cocultured, fine details of their interactions could be revealed in time-lapse experiments. Thus, the method provides a useful tool for elucidation of genes involved in different neuronal functions, including neurite outgrowth, synaptogenesis and synaptic transmission.     

Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons

The development of methodologies for gene transfer into the central nervous system is crucial for gene therapy of neurological disorders. In this study, different cationic liposome formulations were used to transfer DNA into C6 glioma cells and primary hippocampal and cortical neurons by varying the nature of the helper lipid (DOPE, Chol) or a mixture of DOPE and cholesterol (Chol) associated to DOTAP. In addition, the effect of the lipid/DNA (+/-) charge ratio, the association of the ligand transferrin to the lipoplexes, and the stage of differentiation of the primary cells on the levels of transfection activity, transfection efficiency, and duration of gene expression were evaluated. Mechanistic studies were also performed to investigate the route of delivery of the complexes into neurons. Our results indicate that DOTAP:Chol (1:1 mol ratio) was the best formulation to transfer a reporter gene into C6 glioma cells, primary hippocampal neurons, and primary cortical neurons. The use of transferrin-associated lipoplexes resulted in a significant enhancement of transfection activity, as compared to plain lipoplexes, which can be partially attributed to the promotion of their internalization mediated by transferrin. While for hippocampal neurons the levels of luciferase gene expression are very low, for primary cortical neurons the levels of transgene expression are high and relatively stable, although only 4% of the cells has been transfected. The stage of cell differentiation revealed to be critical to the levels of gene expression. Consistent with previous findings on the mechanisms of cell internalization, the experiments with inhibitors of the endocytotic pathway clearly indicate that transferrin-associated lipoplexes are internalized into primary neurons by endocytosis. Promising results were obtained in terms of the levels and duration of gene expression, particularly in cortical neurons when transfected with the Tf-associated lipoplexes, this finding suggesting the usefulness of these lipid-based carriers to deliver genes within the CNS.     

Lack of experience‐dependent intrinsic plasticity in the adolescent infralimbic medial prefrontal cortex

Fear extinction, an inhibitory learning that suppresses a previously learned fear memory, is diminished during adolescence. Earlier studies have shown that this suppressed fear extinction during adolescence involves an altered glutamatergic plasticity in infralimbic medial prefrontal cortical (IL‐mPFC) pyramidal neurons. However, it is unclear whether the excitability of IL‐mPFC pyramidal neurons plays a role in this development‐dependent suppression of fear extinction. Therefore, we examined whether fear conditioning and extinction affect the active and passive membrane properties of IL‐mPFC layer 5 pyramidal neurons in preadolescent, adolescent and adult mice. Both preadolescent and adult mice exhibited a bidirectional modulation of the excitability of IL‐mPFC layer 5 pyramidal neurons following fear conditioning and extinction, i.e., fear conditioning reduced membrane excitability, whereas fear extinction reversed this effect. However, the fear conditioning‐induced suppression of excitability was not reversed in adolescent mice following fear extinction training. Neither fear conditioning nor extinction affected GABAergic transmission in IL‐mPFC layer 5 pyramidal neurons, suggesting that GABAergic transmission did not play a role in experience‐dependent modulation of neuronal excitability. Our results suggest that the extinction‐specific modulation of excitability is impaired during adolescence.     

Temporal interactions in the cat visual system. II. Suppressive and facilitatory effects in the lateral geniculate nucleus

Extracellular responses were recorded from single neurons in the lateral geniculate nucleus (LGN) of the cat during presentation of pairs of brief visual stimuli identical to those that produce orientation-selective paired-pulsed suppression in the visual cortex. LGN neurons also show paired-pulse suppression, but the suppression is not orientation selective, and it occurs only for short interstimulus intervals (ISIs; usually less than 200 msec). At longer ISIs, most LGN neurons show a period of facilitation. Thus, the paired-pulse suppression in the LGN cannot account for that seen in the visual cortex. Paired-pulse suppression in the LGN was found to be enhanced by stimulation of the receptive field surround. LGN neurons also showed a second type of suppression, termed "offset suppression," which consisted of a more long-lasting suppression of spontaneous activity following the offset of an excitatory visual stimulus. The suppression of spontaneous activity was accompanied by a reduction of the antidromic excitability, assessed by stimulating LGN axons within the cortex or optic radiation. Unlike paired-pulsed suppression, offset suppression was not enhanced by increased stimulation of the receptive field surround. Paired-pulse suppression and offset suppression are most likely due to different mechanisms because they have different time courses and depend differently on the spatial properties of the stimuli. Functionally, paired-pulse suppression may be related to the reduced visual sensitivity that accompanies eye movements, while offset suppression may serve to enhance temporal contrast.     

Appearance of NMDA receptors triggered by anoxia independent of voltage in vivo and in vitro

Using rat hippocampus we have studied the pattern of neuronal death, abnormal discharge and loss of electrical excitability in slices prepared from animals subjected to bilateral, four-vessel cerebral anoxia and in slices prepared from normal animals that are subjected to anoxia in the recording chamber. As others have reported, pyramidal neurons in area CA1 are lost first after anoxia, while CA3 neurons have an intermediate sensitivity, and those in dentate are relatively anoxia-resistant. After anoxic damage to the intact animal, neurons in both CA1 and CA3 show abnormal bursting discharges in response to synaptic activation for several days, and then the response in CA1 decreases in amplitude and finally the area become unexcitable. While antagonists for N-methyl-D-aspartate (NMDA) receptors have essentially no effect on synaptic responses in control animals, they reduce the bursting responses and greatly depress the small responses in CA1 as neurons are becoming unexcitable after anoxia. With intracellular recording CA1 neurons from animals made transiently anoxic, in contrast to controls, show prolonged synaptic responses, the later components of which are blocked by NMDA antagonists. When slices from normal animals are subjected to anoxia such that excitability is totally lost over a period of about 10 min, there is no significant membrane depolarization during the anoxic episode and recovery of excitability occurs with reoxygenation. However, a period of hyperexcitability and bursting follows and electrical excitability is lost in CA1 but not CA3 neurons after about 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of prolonged cathodal direct current stimulation on the excitatory and inhibitory circuits of the ipsilateral and contralateral motor cortex

Weak cathodal transcranial direct current stimulation (tDCS) of the human hand area modulates corticospinal excitability with a suppression of motor-evoked potentials (MEPs) evoked by transcranial magnetic stimulation (TMS). The changes in excitability persist beyond the time of stimulation if tDCS is given for several minutes and can remain stable for an hour or more. The aim of present study was to evaluate whether a long-lasting suppression of cortical excitability could be induced by prolonged cathodal tDCS (20?min of stimulation). We also explored the impact of brain-derived neurotrophic factor (BDNF) gene polymorphisms, on tDCS after-effects. Cortical excitability to single and paired-pulse TMS was evaluated both for the stimulated and contralateral hemisphere, before and up to 24?h after 20?min of cathodal tDCS. We evaluated threshold and amplitude of MEPs, short interval intracortical inhibition (SICI), and intracortical facilitation (ICF). tDCS produced a pronounced suppression of MEP amplitude that was still significant at 3?h after the end of stimulation. The BDNF genotype had not influence on tDCS after-effects. Thresholds for MEPs, SICI and ICF were not affected. No significant effect was observed in the contralateral hemisphere. Twenty minutes of cathodal tDCS is capable of inducing a long-lasting suppression of the excitability of the human motor cortex.     

Anticonvulsants suppress c-Fos protein expression in spinal cord neurons following noxious thermal stimulation

The expression of the nuclear immediate-early gene-encoded protein c-Fos in spinal cord dorsal horn neurons of the rat following noxious thermal stimulation was compared in carbamazepine-, valproate- and phenytoine-treated animals. Single intraperitoneal injection of carbamazepine (50 mg/kg), valproate (300 mg/kg) or intravenous injection of phenytoine (20 mg/kg) before noxious stimulation reduced the number of c-Fos immunoreactive neurons to 65–80% of control levels in superficial laminae and to 30–60% in deep laminae of the dorsal horn. Pretreatment with carbamazepine or valproate for 4 or 8 days combined with an injection immediately before noxious stimulation further significantly decreased the number of c-Fos neurons in the deep dorsal horn only in animals treated with valproate. The observation that activity-dependent gene expression in the spinal cord is effectively modulated by anticonvulsants discloses a novel therapeutic potential of these compounds. Presumably via an acute suppression of high-frequency repetitive firing and/or altered synaptic transmission of intraspinal or descending neurotransmitter systems these drugs gain access to neuroplastic mechanisms which might be relevant for the restoration of physiological levels of neuronal excitability in the central nervous system.     

Mouse cytomegalovirus in developing brain tissue: analysis of 11 species with GFP-expressing recombinant virus

Cytomegaloviruses (CMVs) are species-specific large double-stranded DNA viruses. Mouse and human CMVs have a similar morphology, similar gene sequence, and exert similar cellular effects, but the replication of the virus outside its primary host species is limited. This may confer upon CMV certain advantages for expression of foreign genes or cellular labels in brain cells of nonhost species. We examined the ability of recombinant mouse (m)CMV expressing green fluorescent protein (GFP) to serve as a vector for transgene expression in developing neurons and glia outside the normal host species. For comparative purposes, 11 species were examined. Mouse CMV reporter gene expression was particularly strong in the developing brain of its normal host species, mouse, where it replicated in cultures and brain slices, leading to cell death. All mammalian species tested (human, rat, gerbil, hamster, mouse) showed reporter gene expression after mCMV infection. High levels of mCMV infection were also found in chicken central nervous system cells in vitro, and a low level of mCMV expression was found after an initial delay in turtle neurons and glia. No mCMV reporter gene expression was found in frog cells or aplysia neurons or glia or in drosophila or fungal cells. Infection of nonmouse neurons by low concentrations of mCMV led to strong expression of GFP in dendrites and axons with normal morphology. Despite the lack of replication, high doses of mCMV induced morphologic changes in neurons and glia from hamster and rat brain slices, leading to cells rounding up, and to the formation of giant cells consisting of an aggregate of many cells fused together into a syncytium. In contrast, in human hippocampal slices, GFP-expressing cells infected with mCMV had a relatively normal appearance 12 days after inoculation. To determine whether a CMV from another species could serve as a vector for gene transfer, a recombinant human CMV-expressing GFP was used for transgene expression in rat brain cells in vitro. Cytomegaloviruses thus have potential as useful vectors for gene transfer and labeling central nervous system cells, with the actions of CMV being dependent on a number of factors.     

In vivo transfection of lamprey brain neurons by gene gun delivery of DNA

The lamprey has been used extensively in studies of CNS axon regeneration. Progress in determining molecular mechanisms involved in regeneration will require the ability to manipulate expression of target genes or to introduce new genes, but in vivo neuronal transfection has posed difficulties in the mature intact nervous system of vertebrates, including the lamprey. In this paper we report successful transfection of neurons in the brain of living lampreys by means of a hand-held Helios Gene Gun. Particle-mediated ("gene gun") gene transfer has been applied to a variety of cell and tissue types but although it has been used in brain slices and dissociated cultured neurons, to our knowledge it has not been reported as a method for transfection of brain cells in a living animal. Gold particles coated with plasmids containing the gene for the reporter beta-galactosidase were propelled by helium at 150--200 psi toward the exposed floor of the 4th ventricle. Transfected animals were examined by X-gal histochemistry at various recovery times. beta-glactosidase activity was detected as early as 2 days after gene transfer and lasted for at least 6 weeks, the longest time studied. Transgene expression lasted longer in neurons than in glia. The expression product was transported anterogradely into reticulospinal axons and by 6 weeks could be traced into the spinal cord for 8--10 mm caudal to the obex. This raises the possibility of identifying the growth cones of developing or regenerating axons belonging to transfected neurons in functional studies of manipulated genes.     

Enhanced adenoviral gene delivery to motor and dorsal root ganglion neurons following injection into demyelinated peripheral nerves

Injection of viral vectors into peripheral nerves may transfer specific genes into their dorsal root ganglion (DRG) neurons and motoneurons. However, myelin sheaths of peripheral axons block the entry of viral particles into nerves. We studied whether mild, transient peripheral nerve demyelination prior to intraneural viral vector injection would enhance gene transfer to target DRG neurons and motoneurons. The right sciatic nerve of C57BL/6 mice was focally demyelinated with 1% lysolecithin, and the left sciatic nerve was similarly injected with saline (control). Five days after demyelination, 0.5 μl of Ad5‐GFP was injected into both sciatic nerves at the site of previous injection. The effectiveness of gene transfer was evaluated by counting GFP⁺ neurons in the DRGs and ventral horns. After peripheral nerve demyelination, there was a fivefold increase in the number of infected DRG neurons and almost a 15‐fold increase in the number of infected motoneurons compared with the control, nondemyelinated side. Focal demyelination reduced the myelin sheath barrier, allowing greater virus–axon contact. Increased CXADR expression on the demyelinated axons facilitated axoplasmic viral entry. No animals sustained any prolonged neurological deficits. Increased gene delivery into DRG neurons and motoneurons may provide effective treatment for amyotrophic lateral sclerosis, pain, and spinal cord injury. © 2010 Wiley‐Liss, Inc.     

The rostral ventrolateral medulla mediates suppression of the circulatory system by the ventromedial nucleus of the hypothalamus

We recently reported that a train of episodic neural discharges within the ventromedial nucleus of the hypothalamus (VMH) associated with suppression of the circulatory system had been determined by monitoring multiple unit activity (MUA). Abrupt increases in neural activity (MUA volleys; 1 to 4 min in duration) accompanied transient decreases in heart rate (HR) and blood pressure (BP), and showed circadian rhythm, occurring every 15 to 30 min in the light phase but seldom in the dark phase. The present study was aimed to determine if neurons in the vasomotor area of the rostral ventrolateral medulla (RVL) are involved in this VMH-induced cardiovascular suppression. MUAs of the VMH and RVL were monitored simultaneously with HR and BP in urethane-anesthetized rats. In synchrony with each MUA volley in the VMH, spontaneous activity of RVL neurons significantly decreased, as well as HR and BP. These RVL neurons are most likely vasomotor neurons because MUA of the RVL was attenuated by baroreceptor reflex activation, and electrical stimulation of these cells through the MUA recording electrodes produced pressor responses. These data suggest that VMH neurons that show a train of episodic discharges suppress the circulatory system at least in part by inhibiting the excitability of vasomotor neurons in the RVL.     

Regulation of T-type Ca<Superscript>2+</Superscript> channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells

Infection of sensory neurons by herpes simplex virus (HSV)-1 disrupts electrical excitability, altering pain sensory transmission. Because of their low threshold for activation, functional expression of T-type Ca²⁺ channels regulates various cell functions, including neuronal excitability and neuronal communication. In this study, we have tested the effect of HSV-1 infection on the functional expression of T-type Ca²⁺ channels in differentiated ND7-23 sensory-like neurons. Voltage-gated Ca²⁺ currents were measured using whole cell patch clamp recordings in differentiated ND7-23 neurons under various culture conditions. Differentiation of ND7-23 cells evokes a significant increase in T-type Ca²⁺ current densities. Increased T-type Ca²⁺ channel expression promotes the morphological differentiation of ND7-23 cells and triggers a rebound depolarization. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca²⁺ channels from the membrane. HSV-1 evoked reduction in the functional expression of T-type Ca²⁺ channels is mediated by several factors, including decreased expression of Ca_v3.2 T-type Ca²⁺ channel subunits and disruption of endocytic transport. Decreased functional expression of T-type Ca²⁺ channels by HSV-1 infection requires protein synthesis and viral replication, but occurs independently of Egr-1 expression. These findings suggest that infection of neuron-like cells by HSV-1 causes a significant disruption in the expression of T-type Ca²⁺ channels, which can results in morphological and functional changes in electrical excitability.