Intact Central Nervous System Research Articles

Abstract 561: Melanocortin-4 Receptor Signaling Is Required For The Anorexic, Cardiovascular And Antidiabetic Actions Of Leptin In Diabetic Rats.

We previously demonstrated that leptin has powerful antidiabetic actions that are mediated mainly through central nervous system (CNS) effects. In this study we tested the specific importance of melanocortin-4 receptor (MC4R) in mediating leptin’s ability to suppress food intake, to increase blood pressure (BP) and heart rate (HR), and to normalize glucose levels in insulin-dependent diabetes. MC4R knockout (MC4R -/- , n=5) and control wild-type rats (WT, n=4) were implanted with BP telemetry transmitters and an intracerebroventricular (ICV) cannula was inserted into the brain lateral ventricle. After 10 days of recovery, BP and HR were measured 24-hrs/day. After stable baseline measurements for 5 days, a single bolus injection of streptozotocin (STZ) (50 mg/kg, ip) was used to induce insulin-dependent diabetes. Eight days after STZ injection, an osmotic pump was implanted subcutaneously and connected to the ICV cannula to deliver leptin (15 μg/day) for 7 days. At baseline, MC4R -/- rats ate 20% more and were 40% heavier than WT rats. Despite being markedly obese, BP was similar (112±2 vs. 111±2 mmHg) and HR was lower in MC4R -/- rats (320±6 vs. 347±5 bpm). Induction of diabetes increased food intake (30%) and reduced BP (~17 mmHg) and HR (~61 bpm) in WT rats, while food intake, BP and HR were reduced by ~10%, 7 mmHg and 33 bpm in MC4R -/- rats. Leptin treatment for 7 days normalized glucose levels (437±10 to 136±18 mg/dl), reduced food intake (40%), and increased HR (+60 bpm) and BP (+9 mmHg) in WT rats. However, only modest changes in glucose levels (367±16 to 326±23 mg/dl), food intake (5%), HR (+16 bpm) and BP (+4 mmHg) were observed in MC4R -/- rats. These results indicate that intact CNS MC4R signaling is necessary for leptin to exert its chronic anorexic, cardiovascular and antidiabetic actions.

Boc modifies the spectrum of holoprosencephaly in the absence of Gas1 function.

ABSTRACTHoloprosencephaly is a heterogeneous developmental malformation of the central nervous system characterized by impaired forebrain cleavage, midline facial anomalies and wide phenotypic variation. Indeed, microforms represent the mildest manifestation, associated with facial anomalies but an intact central nervous system. In many cases, perturbations in sonic hedgehog signaling are responsible for holoprosencephaly. Here, we have elucidated the contribution of Gas1 and an additional hedgehog co-receptor, Boc during early development of the craniofacial midline, by generating single and compound mutant mice. Significantly, we find Boc has an essential role in the etiology of a unique form of lobar holoprosencephaly that only occurs in conjunction with combined loss of Gas1. Whilst Gas1−/− mice have microform holoprosencephaly characterized by a single median maxillary central incisor, cleft palate and pituitary anomalies, Boc−/− mice have a normal facial midline. However, Gas1−/−; Boc−/− mutants have lobar holoprosencephaly associated with clefting of the lip, palate and tongue, secondary to reduced sonic hedgehog transduction in the central nervous system and face. Moreover, maxillary incisor development is severely disrupted in these mice, arresting prior to cellular differentiation as a result of apoptosis in the odontogenic epithelium. Thus, Boc and Gas1 retain an essential function in these tooth germs, independent of their role in midline development of the central nervous system and face. Collectively, this phenotype demonstrates both redundancy and individual requirements for Gas1 and Boc during sonic hedgehog transduction in the craniofacial midline and suggests BOC as a potential digenic locus for lobar holoprosencephaly in human populations.

Thymic stromal lymphopoietin is expressed in the intact central nervous system and upregulated in the myelin‐degenerative central nervous system

Thymic stromal lymphopoietin (TSLP) is an epithelial cytokine expressed at barrier surfaces of the skin, gut, nose, lung, and the maternal/fetal interphase. At these sites, it is important for the generation and maintenance of non-inflammatory, tissue-resident dendritic cell responses. We show here that TSLP is also expressed in the central nervous system (CNS) where it is produced by choroid plexus epithelial cells and astrocytes in the spinal cord. Under conditions of low-grade myelin degeneration, the numbers of TSLP-expressing astrocytes increase, and microglia express transcripts for the functional TSLP receptor dimer indicating that these cells are targets for TSLP in the myelin-degenerative CNS.

Prosodic expression and levodopa in Parkinson's disease

rosody is an important supra-segmental aspect of speech. Through prosodic varia-tion, many cues regarding message intention, affected expression and type of in-terpersonal relationship are given to the listener. The main characteristics involved in prosody are fundamental frequency and loudness variability, speech rate, dura-tion of pauses, vowel length, force of articulation and rhythm. Prosody pattern is an individual characteristic that requires an intact central and periphe-ral nervous system. It is highly influenced by biological factors, cultural features, personality traits, emotional states and aging. It is easy to understand that many neurological and men-tal disorders can produce a direct impact on prosodic expression. For example, maniacal epi-sodes are vocally characterized by uncontrolled intensity and excessive frequency variability; on the other hand, vocal fold paralysis can severely limit frequency and loudness control, im-pairing the speaking and singing voice. Parkinson’s disease (PD) patients suffer from reduction of the extent of all movements re-lating to speech, involving three subsystems: respiratory, phonatory and articulatory. In addi-tion, swallowing may also be impaired

Nogo-A Represses Anatomical and Synaptic Plasticity in the Central Nervous System

Nogo-A was initially discovered as a myelin-associated growth inhibitory protein limiting axonal regeneration after central nervous system (CNS) injury. This review summarizes current knowledge on how myelin and neuronal Nogo-A and its receptors exert physiological functions ranging from the regulation of growth suppression to synaptic plasticity in the developing and adult intact CNS.

Abstract 3302: Antisense oligonucleotide depletion of the mitotic kinesin Eg5 by direct delivery to the brain could be a useful strategy for treating glioma tumors.

Abstract To date, all approved chemotherapeutic agents which target the mitotic cell division interfere with spindle microtubule dynamics, leading to mitotic arrest and apoptosis. While effective, these drugs are subject to resistance mechanisms and they are also associated with a variety of side effects, including neurotoxicity. Their use in treating nervous system tumors is therefore not warranted. One strategy to target mitosis, without damaging microtubules in non-dividing neurons, would be to inhibit key mitotic components, such as the mitotic kinesin Eg5, which is required for establishing a normal bipolar mitotic spindle. We have shown that glioblastoma cells depleted for Eg5 arrest in the next mitosis. After a prolonged arrest, they may slip out and become multinucleated, which will likely prevent further successful divisions or they may go into apoptosis. Further, mitotic arrest and induction of apoptosis in Eg5 depleted glioblastoma cells occur independent of p53, Rb-signalling and the PI3K-pathway suggesting that Eg5 is a potential therapeutic target for glioblastoma patients with different underlying genetic abnormalities We have also tested the clinical feasibility of using a cell cycle targeting antisense oligonucleotide based therapy delivered directly to the central nervous system (CNS) as a novel treatment for glioblastoma tumors. This work has demonstrated that intraventricular administration of ASOs can efficiently target cells in the CNS and be delivered to glioma-initiating neural stem cells transplanted into the cortex of naïve mice as well as to glioblastoma tumors in a genetically predisposed mouse model. This strategy is therefore a potential route of administration for treating glioblastoma tumors which originate in the CNS. Direct targeting of mitotic components in the brain should have a limited toxicity to non-cycling neurons and as a benefit, as long as the blood-brain barrier is intact direct CNS delivery should have minimal dose-limiting toxicity outside of the CNS. Ongoing studies will determine the effect of Eg5 inhibition on glioblastoma growth in vivo. Citation Format: Cecilia C. Krona, Jihane Boubaker, Dinorah Friedmann-Morvinski, Alex Wong, Melissa McAlonis-Downes, Erich Koller, Aneeza S. Kim, Gene Hung, Frank Rigo, Seung Chun, Benjamin Vitre, Frank Bennett, Inder Verma, Don W. Cleveland. Antisense oligonucleotide depletion of the mitotic kinesin Eg5 by direct delivery to the brain could be a useful strategy for treating glioma tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3302. doi:10.1158/1538-7445.AM2013-3302

A neuroprosthesis for tremor management through the control of muscle co-contraction

BackgroundPathological tremor is the most prevalent movement disorder. Current treatments do not attain a significant tremor reduction in a large proportion of patients, which makes tremor a major cause of loss of quality of life. For instance, according to some estimates, 65% of those suffering from upper limb tremor report serious difficulties during daily living. Therefore, novel forms for tremor management are required. Since muscles intrinsically behave as a low pass filter, and tremor frequency is above that of volitional movements, the authors envisioned the exploitation of these properties as a means of developing a novel treatment alternative. This treatment would rely on muscle co-contraction for tremor management, similarly to the strategy employed by the intact central nervous system to stabilize a limb during certain tasks.MethodsWe implemented a neuroprosthesis that regulated the level of muscle co-contraction by injecting current at a pair of antagonists through transcutaneous neurostimulation. Co-contraction was adapted to the instantaneous parameters of tremor, which were estimated from the raw recordings of a pair of solid state gyroscopes with a purposely designed adaptive algorithm. For the experimental validation, we enrolled six patients suffering from parkinsonian or essential tremor of different severity, and evaluated the effect of the neuroprosthesis during standard tasks employed for neurological examination.ResultsThe neuroprosthesis attained significant attenuation of tremor (p<0.001), and reduced its amplitude up to a 52.33±25.48%. Furthermore, it alleviated both essential and parkinsonian tremor in spite of their different etiology and symptomatology. Tremor severity was not a limiting factor on the performance of the neuroprosthesis, although there was a subtle trend towards larger attenuation of more severe tremors. Tremor frequency was not altered during neurostimulation, as expected from the central origin of Parkinson’s disease and essential tremor. All patients showed a good tolerance to neurostimulation in terms of comfort and absence of pain, and some spontaneously reported that they felt that tremor was reduced when the neuroprosthesis was activated.ConclusionsThe results presented herein demonstrate that the neuroprosthesis provides systematic attenuation of the two major types of tremor, irrespectively from their severity. This study sets the basis for the validation of the neuroprosthesis as an alternative, non-invasive means for tremor management.

The “quad‐partite” synapse: Microglia‐synapse interactions in the developing and mature CNS

Microglia are the resident immune cells and phagocytes of our central nervous system (CNS). While most work has focused on the rapid and robust responses of microglia during CNS disease and injury, emerging evidence suggests that these mysterious cells have important roles at CNS synapses in the healthy, intact CNS. Groundbreaking live imaging studies in the anesthetized, adult mouse demonstrated that microglia processes dynamically survey their environment and interact with other brain cells including neurons and astrocytes. More recent imaging studies have revealed that microglia dynamically interact with synapses where they appear to serve as "synaptic sensors," responding to changes in neural activity and neurotransmitter release. In the following review, we discuss the most recent work demonstrating that microglia play active roles at developing and mature synapses. We first discuss the important imaging studies that have led us to better understand the physical relationship between microglia and synapses in the healthy brain. Following this discussion, we review known molecular mechanisms and functional consequences of microglia-synapse interactions in the developing and mature CNS. Our current knowledge sheds new light on the critical functions of these mysterious cells in synapse development and function in the healthy CNS, but has also incited several new and interesting questions that remain to be explored. We discuss these open questions, and how the most recent findings in the healthy CNS may be related to pathologies associated with abnormal and/or loss of neural circuits.

Repeatability of Corticospinal and Spinal Measures during Lengthening and Shortening Contractions in the Human Tibialis Anterior Muscle

Elements of the human central nervous system (CNS) constantly oscillate. In addition, there are also methodological factors and changes in muscle mechanics during dynamic muscle contractions that threaten the stability and consistency of transcranial magnetic stimulation (TMS) and perpherial nerve stimulation (PNS) measures.PurposeTo determine the repeatability of TMS and PNS measures during lengthening and shortening muscle actions in the intact human tibialis anterior.MethodsOn three consecutive days, 20 males performed lengthening and shortening muscle actions at 15, 25, 50 and 80% of maximal voluntary contraction (MVC). The amplitude of the Motor Evoked Potentials (MEPs) produced by TMS was measured at rest and during muscle contraction at 90° of ankle joint position. MEPs were normalised to Mmax determined with PNS. The corticospinal silent period was recorded at 80% MVC. Hoffman reflex (H-reflex) at 10% isometric and 25% shortening and lengthening MVCs, and V-waves during MVCs were also evoked on each of the three days.ResultsWith the exception of MEPs evoked at 80% shortening MVC, all TMS-derived measures showed good reliability (ICC = 0.81–0.94) from days 2 to 3. Confidence intervals (CI, 95%) were lower between days 2 and 3 when compared to days 1 and 2. MEPs significantly increased at rest from days 1 to 2 (P = 0.016) and days 1 to 3 (P = 0.046). The H-reflex during dynamic muscle contraction was reliable across the three days (ICC = 0.76–0.84). V-waves (shortening, ICC = 0.77, lengthening ICC = 0.54) and the H-reflex at 10% isometric MVC (ICC = 0.66) was generally less reliable over the three days.ConclusionAlthough it is well known that measures of the intact human CNS exhibit moment-to-moment fluctuations, careful experimental arrangements make it possible to obtain consistent and repeatable measurements of corticospinal and spinal excitability in the actively lengthening and shortening human TA muscle.

CD4(+) CD25(+) FoxP3(+) regulatory T cells suppress cytotoxicity of CD8(+) effector T cells: implications for their capacity to limit inflammatory central nervous system damage at the parenchymal level.

BackgroundCD4+ CD25+ forkhead box P3 (FoxP3)+ regulatory T cells (T reg cells) are known to suppress adaptive immune responses, key control tolerance and autoimmunity.MethodsWe challenged the role of CD4+ T reg cells in suppressing established CD8+ T effector cell responses by using the OT-I/II system in vitro and an OT-I-mediated, oligodendrocyte directed ex vivo model (ODC-OVA model).ResultsCD4+ T reg cells dampened cytotoxicity of an ongoing CD8+ T effector cell attack in vitro and within intact central nervous system tissue ex vivo. However, their suppressive effect was limited by the strength of the antigen signal delivered to the CD8+ T effector cells and the ratio of regulatory to effector T cells. CD8+ T effector cell suppression required T cell receptor-mediated activation together with costimulation of CD4+ T reg cells, but following activation, suppression did not require restimulation and was antigen non-specific.ConclusionsOur results suggest that CD4+ T reg cells are capable of suppressing CD8+ T effector cell responses at the parenchymal site, that is, limiting parenchymal damage in autoimmune central nervous system inflammation.

Functional evidence for D‐serine inhibition of non‐N‐methyl‐D‐aspartate ionotropic glutamate receptors in retinal neurons

D-Serine is an important signaling molecule throughout the central nervous system, acting as an N-methyl-D-aspartate (NMDA) receptor coagonist. This study investigated the D-serine modulation of non-NMDA ionotropic glutamate receptors expressed by inner retinal neurons. We first identified that the degradation of endogenous retinal D-serine, by application of D-amino acid oxidase, caused an enhancement of kainate- and α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor-mediated calcium responses from the ganglion cell layer of the isolated rat retina and light-evoked responses obtained by multi-electrode array recordings from the guinea pig retina. Approximately 30-45% of cells were endogenously inhibited by D-serine, as suggested by the effect of D-amino acid oxidase. Conversely, bath application of D-serine caused a reduction in multi-electrode array recorded responses and decreased kainate, but not potassium-induced calcium responses, in a concentration-dependent manner (IC(50), 280 μm). Using cultured retinal ganglion cells to reduce network influences, D-serine reduced kainate-induced calcium responses and AMPA induced whole-cell currents. Finally, the inhibitory effect of D-serine on the kainate-induced calcium response was abolished by IEM 1460, thereby identifying calcium-permeable AMPA receptors as a potential target for D-serine. To our knowledge, this is the first study to address specifically the effect of D-serine on AMPA/kainate receptors in intact central nervous system tissue, to identify its effect on calcium permeable AMPA receptors and to report the endogenous inhibition of AMPA/kainate receptors.

Dissection of Third-Instar Drosophila Larvae for Electrophysiological Recording from Neurons

The fruit fly Drosophila melanogaster has been instrumental in expanding our understanding of early aspects of neural development. The use of this model system has greatly added to our knowledge of neural cell-fate determination, axon guidance, and synapse formation. It has also become possible to access and make electrophysiological recordings directly from neurons in situ in an intact central nervous system (CNS), which has facilitated studies of the development and regulation of neuronal signaling. It is possible to obtain electrophysiological recordings from all stages of Drosophila. Exposure of the intact Drosophila CNS is a prerequisite for such electrophysiological recordings. The dissection procedure described here is suitable for third-instar larvae. The dissection should take ∼5 min to complete if all preparation work has been completed in advance. Owing to the short life span of the dissected larva, it is not recommended that the procedure be stopped or the preparation stored for later use.

Dissection of First- and Second-Instar Drosophila Larvae for Electrophysiological Recording from Neurons: The Flat (or Fillet) Preparation: Figure 1.

The fruit fly Drosophila melanogaster has been instrumental in expanding our understanding of early aspects of neural development. The use of this model system has greatly added to our knowledge of neural cell-fate determination, axon guidance, and synapse formation. It has also become possible to access and make electrophysiological recordings directly from neurons in situ in an intact central nervous system (CNS), which has facilitated studies of the development and regulation of neuronal signaling. It is possible to obtain electrophysiological recordings from all stages of Drosophila. Exposure of the intact Drosophila CNS is a prerequisite for such electrophysiological recordings. The dissection procedure described here can be applied to both late-stage embryos (stage 16 onward) and larvae. Because of their size, third-instar larvae are more difficult to flatten using this method and, if recording from this stage, the reader might consider using insect pins for the dissection or isolating the CNS using an alternative method. The dissection should take <10 min if all preparation work has been completed in advance. Owing to the short life span of the dissected larva, it is not recommended that the procedure be stopped or the preparation stored for later use.

Whole-Cell Patch Recording from Drosophila Larval Neurons

The fruit fly Drosophila melanogaster has been instrumental in expanding our understanding of early aspects of neural development. The use of this model system has greatly added to our knowledge of neural cell-fate determination, axon guidance, and synapse formation. It has also become possible to access and make electrophysiological recordings directly from neurons in situ in an intact central nervous system (CNS), which has facilitated studies of the development and regulation of neuronal signaling. This protocol describes a procedure for revealing larval motor neurons and applying whole-cell patch recording techniques to these cells. The useful lifetime of first-instar larval preparations is ∼30 min, and that of third-instar CNS preparations is up to 1 h. It is therefore recommended that fresh preparations are used and that no breaks are taken during the procedure, although there may be time to pull and polish a patch pipette.

Rapid quantification of inflammation in tissue samples using perfluorocarbon emulsion and fluorine-19 nuclear magnetic resonance.

Quantification of inflammation in tissue samples can be a time-intensive bottleneck in therapeutic discovery and preclinical endeavors. We describe a versatile and rapid approach to quantitatively assay macrophage burden in intact tissue samples. Perfluorocarbon (PFC) emulsion is injected intravenously, and the emulsion droplets are effectively taken up by monocytes and macrophages. These 'in situ' labeled cells participate in inflammatory events in vivo resulting in PFC accumulation at inflammatory loci. Necropsied tissues or intact organs are subjected to conventional fluorine-19 ((19)F) NMR spectroscopy to quantify the total fluorine content per sample, proportional to the macrophage burden. We applied these methods to a rat model of experimental allergic encephalomyelitis (EAE) exhibiting extensive inflammation and demyelination in the central nervous system (CNS), particularly in the spinal cord. In a cohort of EAE rats, we used (19)F NMR to derive an inflammation index (IFI) in intact CNS tissues. Immunohistochemistry was used to confirm intracellular colocalization of the PFC droplets within CNS CD68+ cells having macrophage morphology. The IFI linearly correlated to mRNA levels of CD68 via real-time PCR analysis. This (19)F NMR approach can accelerate tissue analysis by at least an order of magnitude compared with histological approaches.

Neural Plasticity: The Biological Substrate For Neurorehabilitation

Abstract Decades of basic science have clearly demonstrated the capacity of the central nervous system (CNS) to structurally and functionally adapt in response to experience. The field of neurorehabilitation has begun to use this body of work to develop neurobiologically informed therapies that harness the key behavioral and neural signals that drive neural plasticity. The present review describes how neural plasticity supports both learning in the intact CNS and functional improvement in the damaged or diseased CNS. A pragmatic, interdisciplinary definition of neural plasticity is presented that may be used by both clinical and basic scientists studying neurorehabilitation. Furthermore, a description of how neural plasticity may act to drive different neural strategies underlying functional improvement after CNS injury or disease is provided. The understanding of the relationship between these different neural strategies, mechanisms of neural plasticity, and changes in behavior may facilitate the development of novel, more effective rehabilitation interventions.

Nociceptor sensitization in pain pathogenesis.

The incidence of chronic pain is estimated to be 20-25% worldwide. Few patients with chronic pain obtain complete relief from the drugs that are currently available, and more than half report inadequate relief. Underlying the challenge of developing better drugs to manage chronic pain is incomplete understanding of the heterogeneity of mechanisms that contribute to the transition from acute tissue insult to chronic pain and to pain conditions for which the underlying pathology is not apparent. An intact central nervous system (CNS) is required for the conscious perception of pain, and changes in the CNS are clearly evident in chronic pain states. However, the blockage of nociceptive input into the CNS can effectively relieve or markedly attenuate discomfort and pain, revealing the importance of ongoing peripheral input to the maintenance of chronic pain. Accordingly, we focus here on nociceptors: their excitability, their heterogeneity and their role in initiating and maintaining pain.

LPA receptor expression in the central nervous system in health and following injury

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA(1-3)) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA(1) was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA(2) showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA(3) showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA(1-3) are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.

Spike timing dependent plasticity in the intact brain: counteracting spurious spike coincidences.

A computationally rich algorithm of synaptic plasticity has been proposed based on the experimental observation that the sign and amplitude of the change in synaptic weight is dictated by the temporal order and temporal contiguity between pre- and postsynaptic activities. For more than a decade, this spike-timing-dependent plasticity (STDP) has been studied mainly in brain slices of different brain structures and cultured neurons. Although not yet compelling, evidences for the STDP rule in the intact brain, including primary sensory cortices, have been provided lastly. From insects to mammals, the presentation of precisely timed sensory inputs drives synaptic and functional plasticity in the intact central nervous system, with similar timing requirements than the in vitro defined STDP rule. The convergent evolution of this plasticity rule in species belonging to so distant phylogenic groups points to the efficiency of STDP, as a mechanism for modifying synaptic weights, as the basis of activity-dependent development, learning and memory. In spite of the ubiquity of STDP phenomena, a number of significant variations of the rule are observed in different structures, neuronal types and even synapses on the same neuron, as well as between in vitro and in vivo conditions. In addition, the state of the neuronal network, its ongoing activity and the activation of ascending neuromodulatory systems in different behavioral conditions have dramatic consequences on the expression of spike-timing-dependent synaptic plasticity, and should be further explored.

Microelectrode investigation of neuroneal ageing from a single identified neurone

Microelectrode amperometry is uniquely suited for characterising the dynamics of neurotransmitter release, as it offers unparalleled spatial and temporal resolution. We have used carbon fibre microelectrodes to study release of the monoamine neurotransmitter serotonin (5-HT) and the gaseous transmitter nitric oxide (NO) in intact central nervous system of the water snail, Lymnaea stagnalis. Analysis of spontaneous vesicular release of 5-HT and depolarisation-induced release of NO reveals significant differences with ageing that may be associated with changes in protein structure and function.