Nerve Terminal Function Research Articles

Impaired sensory nerve function and axon morphology in mice with diabetic neuropathy

Diabetes is the most prevalent metabolic disorder in the United States, and between 50% and 70% of diabetic patients suffer from diabetes-induced neuropathy. Yet our current knowledge of the functional changes in sensory nerves and their distal terminals caused by diabetes is limited. Here, we set out to investigate the functional and morphological consequences of diabetes on specific subtypes of cutaneous sensory nerves in mice. Diabetes was induced in C57Bl/6 mice by a single intraperitoneal injection of streptozotocin. After 6-8 wk, mice were characterized for behavioral sensitivity to mechanical and heat stimuli followed by analysis of sensory function using teased nerve fiber recordings and histological assessment of nerve fiber morphology. Diabetes produced severe functional impairment of C-fibers and rapidly adapting Aβ-fibers, leading to behavioral hyposensitivity to both mechanical and heat stimuli. Electron microscopy images showed that diabetic nerves have axoplasm with more concentrated organelles and frequent axon-myelin separations compared with control nerves. These changes were restricted to the distal nerve segments nearing their innervation territory. Furthermore, the relative proportion of Aβ-fibers was reduced in diabetic skin-nerve preparations compared with nondiabetic control mice. These data identify significant deficits in sensory nerve terminal function that are associated with distal fiber loss, morphological damage, and behavioral hyposensitivity in diabetic C57Bl/6 mice. These findings suggest that diabetes damages sensory nerves, leading to functional deficits in sensory signaling that underlie the loss of tactile acuity and pain sensation associated with insensate diabetic neuropathy.

Cysteine String Protein-α Prevents Activity-Dependent Degeneration in GABAergic Synapses

The continuous release of neurotransmitter could be seen to place a persistent burden on presynaptic proteins, one that could compromise nerve terminal function. This supposition and the molecular mechanisms that might protect highly active synapses merit investigation. In hippocampal cultures from knock-out mice lacking the presynaptic cochaperone cysteine string protein-alpha (CSP-alpha), we observe progressive degeneration of highly active synaptotagmin 2 (Syt2)-expressing GABAergic synapses, but surprisingly not of glutamatergic terminals. In CSP-alpha knock-out mice, synaptic degeneration of basket cell terminals occurs in vivo in the presence of normal glutamatergic synapses onto dentate gyrus granule cells. Consistent with this, in hippocampal cultures from these mice, the frequency of miniature IPSCs, caused by spontaneous GABA release, progressively declines, whereas the frequency of miniature excitatory AMPA receptor-mediated currents (mEPSCs), caused by spontaneous release of glutamate, is normal. However, the mEPSC amplitude progressively decreases. Remarkably, long-term block of glutamatergic transmission in cultures lacking CSP-alpha substantially rescues Syt2-expressing GABAergic synapses from neurodegeneration. These findings demonstrate that elevated neural activity increases synapse vulnerability and that CSP-alpha is essential to maintain presynaptic function under a physiologically high-activity regimen.

Huntingtin-associated Protein-1 Deficiency in Orexin-producing Neurons Impairs Neuronal Process Extension and Leads to Abnormal Behavior in Mice

Huntingtin-associated protein-1 (Hap1) is a neuronal protein that associates with huntingtin, the Huntington disease protein. Although Hap1 and huntingtin are known to be involved in intracellular trafficking, whether and how the impairment of Hap1-associated trafficking leads to neurological pathology and symptoms remain to be seen. As Hap1 is enriched in neuronal cells in the brain, addressing this issue is important in defining the role of defective intracellular trafficking in the selective neuropathology associated with Hap1 dysfunction. Here, we find that Hap1 is abundantly expressed in orexin (hypocretin)-producing neurons (orexin neurons), which are distinctly distributed in the hypothalamus and play an important role in the regulation of feeding and behavior. We created conditional Hap1 knock-out mice to selectively deplete Hap1 in orexin neurons via the Cre-loxP system. These mice show process fragmentation of orexin neurons and reductions in food intake, body weight, and locomotor activity. Sucrose density gradient fractionation reveals that loss of Hap1 in the mouse brain also reduces the distribution of trafficking protein complexes and cargo proteins in the fractions that are enriched in synaptosomes. These results suggest that Hap1 is critical for the transport of multiple proteins to the nerve terminals to maintain the integrity of neuronal processes and that selective disruption of the processes of orexin neurons can cause abnormal feeding and locomotor activity.

Electrophysiological Identification of the Functional Presynaptic Nerve Terminals on an Isolated Single Vasopressin Neurone of the Rat Supraoptic Nucleus

Release of arginine vasopressin (AVP) and oxytocin from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is under the control of glutamate-dependent excitation and GABA-dependent inhibition. The possible role of the synaptic terminals attached to SON neurones has been investigated using whole-cell patch-clamp recording in in vitro rat brain slice preparations. Recent evidence has provided new insights into the repercussions of glial environment modifications on the physiology of MNCs at the synaptic level in the SON. In the present study, excitatory glutamatergic and inhibitory GABAergic synaptic inputs were recorded from an isolated single SON neurone cultured for 12 h, using the whole-cell patch clamp technique. Neurones expressed an AVP-enhanced green fluorescent protein (eGFP) fusion gene in MNCs. In addition, native synaptic terminals attached to a dissociated AVP-eGFP neurone were visualised with synaptic vesicle markers. These results suggest that the function of presynaptic nerve terminals may be evaluated directly in a single AVP-eGFP neurone. These preparations would be helpful in future studies aiming to electrophysiologically distinguish between the functions of synaptic terminals and glial modifications in the SON neurones.

Erratum: Analysis of gene expression in two large schizophrenia cohorts identifies multiple changes associated with nerve terminal function

Erratum: Analysis of gene expression in two large schizophrenia cohorts identifies multiple changes associated with nerve terminal function

Erratum: Analysis of gene expression in two large schizophrenia cohorts identifies multiple changes associated with nerve terminal function

Erratum: Analysis of gene expression in two large schizophrenia cohorts identifies multiple changes associated with nerve terminal function

Mutation of OPA1 gene causes deafness by affecting function of auditory nerve terminals

Autosomal dominant optic atrophy (DOA) is a retinal neuronal degenerative disease characterized by a progressive bilateral visual loss. We report on two affected members of a family with dominantly inherited neuropathy of both optic and auditory nerves expressed by impaired visual acuity, moderate pure tone hearing loss, and marked loss of speech perception. We investigated cochlear abnormalities accompanying the hearing loss and the effects of cochlear implantation. We sequenced OPA1 gene and recorded cochlear receptor and neural potentials before cochlear implantation. Genetic analysis identified R445H mutation in OPA1 gene. Audiological studies showed preserved cochlear receptor outer hair cell activities (otoacoustic emissions) and absent or abnormally delayed auditory brainstem responses (ABRs). Trans-tympanic electrocochleography (ECochG) showed prolonged low amplitude negative potentials without auditory nerve compound action potentials. The latency of onset of the cochlear potentials was within the normal range found for inner hair cell summating receptor potentials. The duration of the negative potential was reduced to normal during rapid stimulation consistent with adaptation of neural sources generating prolonged cochlear potentials. Both subjects had cochlear implants placed with restoration of hearing thresholds, speech perception, and synchronous activity in auditory brainstem pathways. The results suggest that deafness accompanying this OPA1 mutation is due to altered function of terminal unmyelinated portions of auditory nerve. Electrical stimulation of the cochlea activated proximal myelinated portions of auditory nerve to restore hearing.

Activity-Dependent Bulk Synaptic Vesicle Endocytosis—A Fast, High Capacity Membrane Retrieval Mechanism

Central nerve terminals are placed under considerable stress during intense stimulation due to large numbers of synaptic vesicles (SVs) fusing with the plasma membrane. Classical clathrin-dependent SV endocytosis cannot correct for the large increase in nerve terminal surface area in the short term, due to its slow kinetics and low capacity. During such intense stimulation, an additional SV retrieval pathway is recruited called bulk endocytosis. Recent studies have shown that bulk endocytosis fulfils all of the physiological requirements to remedy the acute changes in nerve terminal surface area to allow the nerve terminal to continue to function. This review will summarise the recent developments in the field that characterise the physiology of bulk endocytosis which show that it is a fast, activity-dependent and high capacity mechanism that is essential for the function of central nerve terminals.

Analysis of gene expression in two large schizophrenia cohorts identifies multiple changes associated with nerve terminal function

Schizophrenia is a severe psychiatric disorder with a world-wide prevalence of 1%. The pathophysiology of the illness is not understood, but is thought to have a strong genetic component with some environmental influences on aetiology. To gain further insight into disease mechanism, we used microarray technology to determine the expression of over 30 000 mRNA transcripts in post-mortem tissue from a brain region associated with the pathophysiology of the disease (Brodmann area 10: anterior prefrontal cortex) in 28 schizophrenic and 23 control patients. We then compared our study (Charing Cross Hospital prospective collection) with that of an independent prefrontal cortex dataset from the Harvard Brain Bank. We report the first direct comparison between two independent studies. A total of 51 gene expression changes have been identified that are common between the schizophrenia cohorts, and 49 show the same direction of disease-associated regulation. In particular, changes were observed in gene sets associated with synaptic vesicle recycling, transmitter release and cytoskeletal dynamics. This strongly suggests multiple, small but synergistic changes in gene expression that affect nerve terminal function.

The human immunodeficiency virus-1–associated protein, Tat 1-86, impairs dopamine transporters and interacts with cocaine to reduce nerve terminal function: A no-net-flux microdialysis study

Injection drug use accounts for approximately one-third of human immunodeficiency virus (HIV) infections in the United States. HIV-associated proteins have been shown to interact with various drugs of abuse to incite concerted neurotoxicity. One common area for their interaction is the nerve terminal, including dopamine transporter (DAT) systems. However, results regarding DAT function and regulation in HIV-infection, regardless of drug use, are mixed. Thus, the present experiments were designed to explicitly control Tat and cocaine administration in an in vivo rat model in order to reconcile differences that exist in the literature to date. We examined Tat plus cocaine-induced alterations using no-net-flux microdialysis, which is sensitive to alterations in DAT function, in order to test the potential for DAT as an early mediator of HIV-induced oxidative stress and neurodegeneration in vivo. Within 5 h of intra-accumbal administration of the HIV-associated protein, Tat, we noted a significant reduction in local DAT efficiency with little change in DA overflow/release dynamics. Further, at 48 h post-Tat administration, we demonstrated a concerted effect of the HIV-protein Tat with cocaine on both uptake and release function. Finally, we discuss the extent to which DAT dysfunction may be considered a predecessor to generalized nerve terminal dysfunction. Characterization of DAT dysfunction in vivo may provide an early pharmacotherapeutic target, which in turn may prevent or attenuate downstream mediators of neurotoxicity (i.e., reactive species) to dopamine systems occurring in neuro-AIDS.

Do nerve terminal sprouts contribute to functional recovery from botulinum neurotoxin A?

Botulinum neurotoxin A (BoNT/A) cleaves SNAP-25, a key protein for exocytosis, and thus blocks the release of the neurotransmitter acetylcholine from the motor nerve terminal at the neuromuscular junction (NMJ) (Schiavo et al. 2000). Despite its high toxicity, BoNT/A (Botox®, Allergan Inc.) in small doses has clinical applications in inhibiting undesirable muscular spasticity and in reducing facial wrinkling (Osborne et al. 2007). In addition to muscle paralysis, BoNT/A induces profuse sprouting of motor nerve terminals (Brown et al. 1981). Whether and how these nerve sprouts may affect the treatment is not clear. Previous studies suggest that the nerve sprouts play a major role in the initial functional recovery from the BoNT/A treatment (Angaut-Petit et al. 1990; de Paiva et al. 1999). However, whether synaptic transmission indeed occurs at the sprouts has not been directly demonstrated. In this issue of The Journal of Physiology, Rogozhin et al. (2008) combined electrophysiological recording with imaging approaches, and found that the original synaptic sites play the predominant role in functional restoration from BoNT/A, rather than the nerve sprouts as previously thought. Taking advantage of a thin muscle (epitrochleoanconeus) and a vital nerve terminal dye, 4-Di-2-ASP (Magrassi et al. 1987), Rogozhin et al. (2008) were able to position extracellular electrodes precisely over individual synaptic boutons at the mouse NMJ for focal recordings of synaptic events (del Castillo & Katz, 1956). Because of its fine spatial resolution of 5–10 μm, the focal recording allows comparisons of synaptic efficacy between the original NMJ and the new synaptic site induced by a single BoNT/A injection. By combining the focal recording with intracellular recording, Rogozhin et al. (2008) estimated that the mean quantal content at the new synaptic site was about 17% of that from the original junctional site during the period of 2–12 weeks following a single injection of BoNT/A. The finding by Rogozhin et al. (2008) of the relatively minor role of new sprouts in functional restoration from BoNT/A is in sharp contrast to the prevailing thinking based primarily on a study by de Paiva et al. (1999). To assess nerve terminal function, de Paiva et al. (1999) labelled motor nerve terminals with FM1-43, a fluorescent dye that binds endocytic synaptic vesicles in an activity-dependent manner (Cochilla et al. 1999). They found much brighter FM1-43 staining in the sprouts than in the original synaptic sites during the initial phase of functional recovery (e.g. 28 days) from BoNT/A, but the opposite was the case at the later phase (e.g. 91 days). Although the brighter FM1-43 staining indicates a stronger release efficacy at the nerve sprouts, it may not effectively translate into a larger postsynaptic potential since only a small fraction of the total length of the nerve sprouts is associated with clusters of acetylcholine receptors (AChRs), which are in general smaller and poorly defined. Indeed, Rogozhin et al. (2008) found that the total area of AChRs associated with the nerve sprouts was only about 30% of that associated with the original area during most of the recovery phase. Thus, while the sprouts are highly capable of vesicle recycling, their role in functional recovery is limited. The findings of Rogozhin et al. (2008) raise several important questions regarding synaptic plasticity at the NMJ. For example, why are only a small fraction of the nerve terminal sprouts associated with AChR clusters? What is the role, if any, of nerve sprouts in synaptic repair? Could the vesicle recycling seen at nerve sprouts result in increased uptakes of the neurotrophic factors essential for motoneuron survival? What mechanisms allow nerve terminals to sprout when vesicle exocytosis and subsequent membrane fusions are blocked by BoNT/A? Why do the nerve sprouts and other structure abnormalities at the NMJ persist after functional recovery from multiple injections of BoNT/A as shown in Rogozhin et al. (2008)? Given that sprouting of nerve terminals and the associated Schwann cells is a common form of synaptic plasticity following the blockade of pre- or postsynaptic activity, as well as by partial denervation (Brown et al. 1981; Kang et al. 2003), the study by Rogozhin et al. (2008) is likely to help address these questions to better understand the mechanisms of synaptic plasticity.

Role of DAT‐SPECT in diagnostic work‐up of Parkinsonism

The diagnosis of idiopathic Parkinson's disease (PD) can be achieved with high degrees of accuracy in cases with full expression of classical clinical features. However, diagnostic uncertainty remains in early disease with subtle or ambiguous signs. Functional imaging has been suggested to increase the diagnostic yield in parkinsonian syndromes with uncertain clinical classification. Loss of striatal dopamine nerve terminal function, a hallmark of neurodegenerative Parkinsonism, is strongly related to decreases of dopamine transporter (DAT) density, which can be measured by single photon emission computed tomography (SPECT). The use of DAT-SPECT facilitates the differential diagnosis in patients with isolated tremor symptoms not fulfilling PD or essential tremor criteria, drug-induced, psychogenic and vascular Parkinsonism as well as dementia when associated with Parkinsonism. This review addresses the value of DAT-SPECT in early differential diagnosis, and its potential as a screening tool for subjects at risk of developing PD as well as issues around the assessment of disease progression. (c) 2007 Movement Disorder Society

Cardiac sympathetic nerve terminal function in congestive heart failure

Increased cardiac release of norepinephrine (NE) and depleted cardiac stores of NE are two salient features of the human failing heart. Researches from my laboratory have shown that these changes are accompanied by a functional defect of NE uptake in the cardiac sympathetic nerve terminals. Our studies have shown that the decrease of NE uptake is caused by reduction of NE transporter density in the sympathetic nerve endings, and this change is responsible, at least in part, for the increased myocardial interstitial NE, decreased myocardial adrenoceptor density, and increased myocyte apoptosis in experimental cardiomyopathies. We have also provided evidence in both intact animals and cultured PC12 cells that the decrease of NE transporter is induced by the actions of oxidative metabolites of exogenous NE, involving endoplasmic reticulum stress and impaired N-glycosylation of the NE transporter. This change in the cardiac sympathetic NE uptake function, as demonstrated by [123I] metaiodobenzylguanidine in human studies, may not only serve as an important prognostic variable in patients with congestive heart failure, but also be used as a surrogate for the efficacies of various therapeutic interventions for heart failure. Finally, increasing evidence suggests and further studies are needed to show that the cardiac sympathetic nerve terminal function may be a direct target for pharmacologic treatment of congestive heart failure.

Role of DAT‐SPECT in the diagnostic work up of Parkinsonism

The diagnosis of idiopathic Parkinson's disease (PD) can be achieved with high degrees of accuracy in cases with full expression of classical clinical features. However, diagnostic uncertainty remains in early disease with subtle or ambiguous signs. Functional imaging has been suggested to increase the diagnostic yield in parkinsonian syndromes with uncertain clinical classification. Loss of striatal dopamine nerve terminal function, a hallmark of neurodegenerative parkinsonism, is strongly related to decreases of dopamine transporter (DAT) density, which can be measured by single photon emission computed tomography (SPECT). The use of DAT-SPECT facilitates the differential diagnosis in patients with isolated tremor symptoms not fulfilling PD or essential tremor criteria, drug-induced, psychogenic and vascular parkinsonism as well as dementia when associated with parkinsonism. This review addresses the value of DAT-SPECT in early differential diagnosis, and its potential as a screening tool for subjects at risk of developing PD as well as issues around the assessment of disease progression.

Differential Loss of Presynaptic Dopaminergic Markers in Parkinsonian Monkeys

Assessment of dopamine nerve terminal function and integrity is a strategy employed to monitor deficits in Parkinson's disease (PD) patients and in preclinical models of PD. Dopamine replacement therapies effectively replenish the diminished supply of endogenous dopamine and provide symptomatic benefit to patients. Tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), and amino acid decarboxylase (AADC) are widely used markers of dopaminergic neurons and terminals. The present studies were initiated to: (a) assess alterations in all four markers in the MPTP primate model of dopaminergic degeneration and (b) to determine whether L-DOPA treatment may itself modulate the expression of these markers. MPTP treatment induced a significant decline of dopaminergic immunoreactive fiber and terminal density in the basal ganglia. The amount of reduction varied between markers. The rank order of presynaptic marker loss, from most to least profound reduction, was TH > VMAT2 > DAT > AADC. Semiquantitative image analysis of relative dopaminergic presynaptic fiber and terminal density illustrated region-specific reduction of all four markers. Double immunofluorescence colocalization of two presynaptic markers on the same tissue section confirmed there was a more dramatic loss of TH than of VMAT2 or of DAT following MPTP treatment. L-DOPA treatment was associated with a significantly higher level of AADC and VMAT2 immunoreactivity in the caudate nucleus compared to placebo. These results illustrate that neurotoxic injury of the dopamine system in primates leads to altered and differential expression of presynaptic dopaminergic markers in the basal ganglia and that expression of such markers may be modulated by L-DOPA therapy. These findings have implications for the use of biomarkers of disease progression as well as for the assessment of neurorestorative strategies, such as cell replacement, for the treatment of PD.

Innervation and connexin isoform expression during diabetes-related bladder dysfunction: early structural vs. neuronal remodelling

Urinary bladder dysfunction is a major complication in diabetes mellitus and its mechanism has been attributed to altered neurological function (autonomic and/or peripheral neuropathy). Previous studies have demonstrated impaired nerve deficiencies, including either loss of nerve function and/or anatomical loss of neuromuscular nerve terminals. While the phenomenon of diabetes-related neurological injury is well recognised, its pathogenesis is not well understood. Using a well established rat model of diabetes (streptozotocin model), we investigated the prevalence of sympathetic and parasympathetic nerves and relative prevalence of connexin isoforms (gap junction proteins) during diabetes-related bladder dysfunction. Immunohistochemistry and digital image analysis was used to detect the prevalence of postsynaptic neuronal markers, NOS1 and connexin isoform expressions. Immunohistochemistry showed significant increases in tyrosine hydroxylase (marker of sympathetic innervation) and decreased vesicular acetylcholine transporter (marker of parasympathetic innervation), predominantly in the smooth muscle layer, 3 days after diabetes induction, when compared to age-matched controls. Time-dependent and cell-specific decreases in the connexin 43 isoform, but transient increases in connexin 32 and 26, were also observed in diabetic rats vs. controls (p<0.05). These data suggest that selective and time-dependent expression of gap junction proteins and altered prevalence of sympathetic/parasympathetic innervation are early events during diabetes-related bladder dysfunction and remodelling.

Overexpression of GD1a Ganglioside Sensitizes Motor Nerve Terminals to Anti-GD1a Antibody-Mediated Injury in a Model of Acute Motor Axonal Neuropathy

Anti-GD1a ganglioside antibodies (Abs) are the serological hallmark of the acute motor axonal form of the post-infectious paralysis, Guillain-Barre syndrome. Development of a disease model in mice has been impeded by the weak immunogenicity of gangliosides and the apparent resistance of GD1a-containing neural membranes to anti-GD1a antibody-mediated injury. Here we used mice with altered ganglioside biosynthesis to generate such a model at motor nerve terminals. First, we bypassed immunological tolerance by immunizing GD1a-deficient, beta-1,4-N-acetylgalactosaminyl transferase knock-out mice with GD1a ganglioside-mimicking antigens from Campylobacter jejuni and generated high-titer anti-GD1a antisera and complement fixing monoclonal Abs (mAbs). Next, we exposed ex vivo nerve-muscle preparations from GD1a-overexpressing, GD3 synthase knock-out mice to the anti-GD1a mAbs in the presence of a source of complement and investigated morphological and electrophysiological damage. Dense antibody and complement deposits were observed only over presynaptic motor axons, accompanied by severe ultrastructural damage and electrophysiological blockade of motor nerve terminal function. Perisynaptic Schwann cells and postsynaptic membranes were unaffected. In contrast, normal mice were not only unresponsive to immunization with GD1a but also resistant to neural injury during anti-GD1a Ab exposure, demonstrating the central role of membrane antigen density in modulating both immune tolerance to GD1a and axonal susceptibility to anti-GD1a Abmediated injury. Identical paralyzing effects were observed when testing mouse and human anti-GD1a-positive sera. These data indicate that anti-GD1a Abs arise via molecular mimicry and are likely to be clinically relevant in injuring peripheral nerve axonal membranes containing sufficiently high levels of GD1a.

Anti-disialoside antibodies kill perisynaptic Schwann cells and damage motor nerve terminals via membrane attack complex in a murine model of neuropathy.

Anti-disialoside antibodies (Abs) that bind NeuAc(alpha2-8) NeuAc epitopes on GQ1b and related gangliosides are found in human autoimmune neuropathy sera and are considered to be pathogenic. In a model system in mice, one mechanism by which anti-disialoside Abs have been demonstrated to induce paralysis is through a complement dependent blocking effect on transmitter release at the neuromuscular junction, similar to the effects of alpha-latrotoxin. Although direct targeting of presynaptic neuronal membranes occurs in this model, concomitant injury to perisynaptic Schwann cells (pSC) could indirectly contribute to this paralytic effect by influencing nerve terminal function and survival. To examine this possibility and the specific complement components that might mediate these effects, we exposed neuromuscular junctions in vivo and in vitro to an anti-disialoside Ab in conjunction with intact and selectively deficient complement sources. Using immuno-electron microscopy, we observed Ab deposits equally distributed on both neuronal and pSC membranes, and ultrastructural evidence of injury at both sites. Presynaptic neuronal injury was demonstrated functionally with microelectrode recordings and histologically as neurofilament loss. As hypothesized, concomitant pSC injury occurred, as indicated by abnormal uptake of ethidium dimer into pSC nuclei. The pSC and nerve terminal damage indicators correlated well with deposition of the pore-forming terminal complement component, membrane attack complex (MAC) in pSC and nerve terminal membranes. Furthermore, both neuronal and pSC injury were exacerbated in tissues from mice lacking the inhibitory complement regulator, CD59, where MAC formation is increased. These data demonstrate that both presynaptic neuronal membranes and pSCs are targets for anti-disialoside Abs, and that the injury to both sites is mediated by MAC and further regulated by CD59. This is the first demonstration that complement mediated pSC injury occurs in a model of autoimmune neuropathy and provides a rationale for investigating the possibility of pSC injury in equivalent conditions in man.

The Effects of rAAV2-Mediated NGF Gene Delivery in Adult and Aged Rats

Nerve growth factor (NGF) therapy has been proposed to treat patients with age-related cognitive deficits, including those with Alzheimer's disease. One promising approach to delivering this protein into brain involves viral vectors. However, little is known about the effects of aging on gene transfer in brain generally and in particular its effect on transgenic NGF expression. To examine the transgene expression and biological effects of NGF gene transfer in adult and aged rats, we delivered mouse NGF with C-terminal myc-tag, using a recombinant adeno-associated virus serotype 2 (rAAV2) vector, into the septum of 6- and 21-month-old Fischer 344/Brown Norway hybrid rats. Other animals received a control vector encoding green fluorescent protein. As expected, this strain of rat demonstrated very few age-related deficits in spatial memory-related behavior in the Morris water task either before gene transfer (6 vs 21 months) or afterward (up to 11 vs 26 months). We found that rAAV2 vectors drove transgene expression in aged rats up to 5 months, although the level of transgene expression was lower than that of adult animals. We also showed that NGF gene transfer into the septum of aged animals induced local trophic effects by increasing the number and soma area of septal cholinergic neurons and improved distal synaptic activity by increasing the level of depolarization-induced acetylcholine (ACh) release from hippocampal synaptic terminals. Interestingly, NGF gene transfer suppressed depolarization-induced ACh release in adult rats. These findings show for the first time, to our knowledge, that septal NGF gene transfer modulates hippocampal nerve terminal function. These results are relevant for the potential clinical application of NGF gene therapy.

In Vivo and In Vitro Effects of Acrylamide on Synaptosomal Neurotransmitter Uptake and Release

Evidence suggests acrylamide (ACR) neurotoxicity is mediated by impaired presynaptic transmission. To assess the effects of ACR on nerve terminal function, [ 3H ]glutamate release and uptake were determined in brain synaptosomes isolated from intoxicated rats (50 mg/kg per day×8 days, i.p. or 21 mg/kg per day×21 days, p.o.). Regardless of ACR dose-rate, a significant reduction in synaptosomal K +-stimulated, Ca 2+-dependent release was detected, whereas kinetic analysis of Na +-dependent uptake did not reveal consistent changes. Immunoblot analysis showed normal protein levels (e.g. SNAP-25) in dysfunctional synaptosomes isolated from ACR-intoxicated rats. This suggests that defective release does not involve changes in protein synthesis and/or anterograde delivery of presynaptic constituents. To identify potential targets, synaptosomes were exposed in vitro to [ 14 C]-ACR and radiolabeled proteins were separated by gel electrophoresis and detected by autoradiography. [ 14 C]-ACR labeling of distinct synaptosomal protein bands (10.5–154,000 kDa) was blocked by the sulfhydryl alkylating agent, N-ethylmaleimide (NEM; 4 mM) but not by the non-neurotoxic structural analog propionamide (10 mM). In vitro characterization of synaptosomal [ 3H ]glutamate uptake and release showed that ACR, NEM and iodoacetic acid (IAA) produced concentration-dependent decreases in each parameter that were highly correlated to reductions in free sulfhydryl content. All three chemicals were equiefficacious with respect to reducing sulfhydryl content and neurotransmitter uptake/release, although the relative potencies differed; NEM> IAA> ACR. Kinetic analysis of uptake showed that in vitro exposure to ACR, IAA or NEM at their respective IC 50’s caused similar reductions in V max. These data suggest that ACR-induced synaptic dysfunction involves adduction of presynaptic protein thiol groups and subsequent reduction in neurotransmitter release.