Serotonergic Fibers Research Articles

The Nature of 3, 4-Methylenedioxymethamphetamine (MDMA)-Induced Serotonergic Dysfunction: Evidence for and Against the Neurodegeneration Hypothesis

High doses of the recreational drug 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) have been well-documented to reduce the expression of serotonergic markers in several forebrain regions of rats and nonhuman primates. Neuroimaging studies further suggest that at least one of these markers, the plasma membrane serotonin transporter (SERT), may also be reduced in heavy Ecstasy users. Such effects, particularly when observed in experimental animal models, have generally been interpreted as reflecting a loss of serotonergic fibers and terminals following MDMA exposure. This view has been challenged, however, based on the finding that MDMA usually does not elicit glial cell reactions known to occur in response to central nervous system (CNS) damage. The aim of this review is to address both sides of the MDMA-neurotoxicity controversy, including recent findings from our laboratory regarding the potential of MDMA to induce serotonergic damage in a rat binge model. Our data add to the growing literature implicating neuroregulatory mechanisms underlying MDMA-induced serotonergic dysfunction and questioning the need to invoke a degenerative response to explain such dysfunction.

Locomotor-activated neurons of the cat. II. Noradrenergic innervation and colocalization with NEα1a or NEα2b receptors in the thoraco-lumbar spinal cord

Norepinephrine (NE) is a strong modulator and/or activator of spinal locomotor networks. Thus noradrenergic fibers likely contact neurons involved in generating locomotion. The aim of the present study was to investigate the noradrenergic innervation of functionally related, locomotor-activated neurons within the thoraco-lumbar spinal cord. This was accomplished by immunohistochemical colocalization of noradrenergic fibers using dopamine-β-hydroxylase or NEα(1A) and NEα(2B) receptors with cells expressing the c-fos gene activity-dependent marker Fos. Experiments were performed on paralyzed, precollicular-postmamillary decerebrate cats, in which locomotion was induced by electrical stimulation of the mesencephalic locomotor region. The majority of Fos labeled neurons, especially abundant in laminae VII and VIII throughout the thoraco-lumbar (T13-L7) region of locomotor animals, showed close contacts with multiple noradrenergic boutons. A small percentage (10-40%) of Fos neurons in the T7-L7 segments showed colocalization with NEα(1A) receptors. In contrast, NEα(2B) receptor immunoreactivity was observed in 70-90% of Fos cells, with no obvious rostrocaudal gradient. In comparison with results obtained from our previous study on the same animals, a significantly smaller proportion of Fos labeled neurons were innervated by noradrenergic than serotonergic fibers, with significant differences observed for laminae VII and VIII in some segments. In lamina VII of the lumbar segments, the degree of monoaminergic receptor subtype/Fos colocalization examined statistically generally fell into the following order: NEα(2B) = 5-HT(2A) ≥ 5-HT(7) = 5-HT(1A) > NEα(1A). These results suggest that noradrenergic modulation of locomotion involves NEα(1A)/NEα(2B) receptors on noradrenergic-innervated locomotor-activated neurons within laminae VII and VIII of thoraco-lumbar segments. Further study of the functional role of these receptors in locomotion is warranted.

Dorsal vs Ventral Hippocampal Neurogenesis: Implications for Cognition and Mood

An emerging view of the hippocampus is that of a functionally heterogeneous structure along its longitudinal axis. Lesion studies reveal that the dorsal (septal pole) hippocampus is involved in learning and spatial memory, whereas the ventral (temporal pole) hippocampus regulates emotional and motivated behaviors (Fanselow and Dong, 2010). Anatomical connectivity and gene expression analyses support this functional dissociation. For example, serotonergic fibers provide denser input to the ventral hippocampus with a concomitant enrichment of 5-HT1A and 2C receptors ventrally (KF Tanaka and R Hen, unpublished). Efferent connectivity indicates that ventral hippocampus can modulate reward circuitry and emotional behavior through projections to nucleus accumbens, prefrontal cortex and amygdala, and stress responses by regulating the hypothalamic–pituitary–adrenal axis (Sahay and Hen, 2007). In both regions, the subgranular zone of the dentate gyrus (DG) continues to produce new neurons in adulthood. These adult-born granule cells (GCs) functionally integrate into the DG circuit, exhibit enhanced excitability, and have a significant impact on both learning and emotional behavior (Sahay and Hen, 2007). As adult neurogenesis has been implicated in both learning and mood, an exciting possibility is that adult-born GCs in the dorsal and ventral hippocampus may be functionally dissociated. Chronic antidepressant treatment increases neurogenesis in the DG, a requirement for some of their behavioral effects (Santarelli et al, 2003). Recent studies suggest that antidepressants regulate behavior by selectively increasing ventral hippocampal neurogenesis. Chronic treatment with agomelatine, a melatonin receptor agonist, and 5-HT2C receptor antagonist with robust effects in animal models as well as efficacy in human major depressive disorder increases neurogenesis selectively in the ventral DG (Banasr et al, 2006). In humans, selective serotonin reuptake inhibitors and tricyclic antidepressants increase proliferating and neuronal precursor cells more prominently in the anterior portion of the DG of patients with MDD as compared to controls and untreated MDD subjects (Boldrini et al, 2009). These two studies, although correlational, provide exciting preliminary evidence that warrants future studies aimed at selectively blocking or stimulating neurogenesis in the ventral hippocampus. In the cognitive realm, adult neurogenesis has recently been implicated in pattern separation, the ability to distinguish between similar contexts (Deng et al, 2010). Owing to their unique physiological properties, adult-born GCs may contribute to pattern separation by modulation of sparse coding in the DG. A recent report indicates that ablation of adult-born GCs increases the magnitude of spontaneous gamma bursts in the dorsal DG and enhances modulation of single unit firing by these bursts (Lacefield et al, 2010). This increase in spontaneous activity suggests that adult-born GCs may modulate network inhibition of mature GCs through either feedback inhibition or synaptic competition. Thus, young GCs may contribute to sparse coding and pattern separation by modulating inhibitory control of the mature GCs in the DG. Deficits in pattern separation may not only impact cognition, but may also contribute to anxiety disorders by impairing the ability to discriminate between safe and fearful contexts. The resulting inability to respond appropriately to similar situations that have differing emotional valence may lead to the generalization observed in certain anxiety disorders such as PTSD. As adult-born GCs have a role in sparse coding in the DG and pattern separation, it will be of great interest to examine how blocking or stimulating ventral hippocampal neurogenesis may influence emotional behavior by modulating pattern separation.

Ultrastructural characterization of tryptophan hydroxylase 2-specific cortical serotonergic fibers and dorsal raphe neuronal cell bodies after MDMA treatment in rat

3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") is a widely used recreational drug known to cause selective long-term serotonergic damage. The aim of this study was to characterize the ultrastructure of serotonergic pericarya and proximal neurites in the dorsal raphe nucleus as well as the ultrastructure of serotonergic axons in the frontal cortex of adolescent Dark Agouti rats 3 days after treatment with 15 mg/kg i.p. MDMA. Light microscopic immunohistochemistry and pre-embedding immunoelectron microscopy with a novel tryptophan hydroxylase-2 (Tph2) specific antibody, as a marker of serotonergic structures. Light microscopic analysis showed reduced serotonergic axon density and aberrant swollen varicosities in the frontal cortex of MDMA-treated animals. According to the electron microscopic analysis, Tph2 exhibited diffuse cytoplasmic immunolocalization in dorsal raphe neuronal cell bodies. The ultrastructural-morphometric analysis of these cell bodies did not indicate pathological changes or significant alteration in the cross-sectional areal density of any examined organelles. Proximal serotonergic neurites in the dorsal raphe exhibited no ultrastructural alteration. However, in the frontal cortex among intact fibers, numerous serotonergic axons with destructed microtubules were found. Most of their mitochondria were intact, albeit some injured axons also contained degenerating mitochondria; moreover, a few of them comprised confluent membrane whorls only. Our treatment protocol does not lead to ultrastructural alteration in the serotonergic dorsal raphe cell bodies and in their proximal neurites but causes impairment in cortical serotonergic axons. In these, the main ultrastructural alteration is the destruction of microtubules although a smaller portion of these axons probably undergo an irreversible damage.

Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) Contributes to Secondary Damage after Spinal Cord Injury

The inflammatory response contributes importantly to secondary tissue damage and functional deficits after spinal cord injury (SCI). In this work, we identified mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2 or MK2), a downstream substrate of p38 MAPK, as a potential target using microarray analysis of contused spinal cord tissue taken at the peak of the inflammatory response. There was increased expression and phosphorylation of MK2 after SCI, with phospho-MK2 expressed in microglia/macrophages, neurons and astrocytes. We examined the role of MK2 in spinal cord contusion injury using MK2(-/-) mice. These results show that locomotor recovery was significantly improved in MK2(-/-) mice, compared with wild-type controls. MK2(-/-) mice showed reduced neuron and myelin loss, and increased sparing of serotonergic fibers in the ventral horn caudal to the injury site. We also found differential expression of matrix metalloproteinase-2 and 9 in MK2(-/-) and wild-type mice after SCI. Significant reduction was also seen in the expression of proinflammatory cytokines and protein nitrosylation in the injured spinal cord of MK2(-/-) mice. Our previous work has shown that macrophages lacking MK2 have an anti-inflammatory phenotype. We now show that there is no difference in the number of macrophages in the injured spinal cord between the two mouse strains and little if any difference in their phagocytic capacity, suggesting that macrophages lacking MK2 have a beneficial phenotype. These findings suggest that a lack of MK2 can reduce tissue damage after SCI and improve locomotor recovery. MK2 may therefore be a useful target to treat acute SCI.

Docosahexaenoic Acid Prevents White Matter Damage after Spinal Cord Injury

We have previously shown that the omega-3 fatty acid docosahexaenoic acid (DHA) significantly improves several histological and behavioral measures after spinal cord injury (SCI). White matter damage plays a key role in neurological outcome following SCI. Therefore, we examined the effects of the acute intravenous (IV) administration of DHA (250 nmol/kg) 30 min after thoracic compression SCI in rats, alone or in combination with a DHA-enriched diet (400 mg/kg/d, administered for 6 weeks post-injury), on white matter pathology. By 1 week post-injury, the acute IV DHA injection led to significantly reduced axonal dysfunction, as indicated by accumulation of β-amyloid precursor protein (-55% compared to vehicle-injected controls) in the dorsal columns. The loss of cytoskeletal proteins following SCI was also significantly reduced. There were 43% and 73% more axons immunoreactive for non-phosphorylated 200-kD neurofilament in the ventral white matter and ventrolateral white matter, respectively, in animals receiving DHA injections than vehicle-injected rats. The acute DHA treatment also led to a significant improvement in microtubule-associated protein-2 immunoreactivity. By 6 weeks, damage to myelin and serotonergic fibers was also reduced. For some of the parameters measured, the combination of DHA injection and DHA-enriched diet led to greater neuroprotection than DHA injection alone. These findings demonstrate the therapeutic potential of DHA in SCI, and clearly indicate that this fatty acid confers significant protection to the white matter.

Self‐assembling peptide amphiphile promotes plasticity of serotonergic fibers following spinal cord injury

Injection into the injured spinal cord of peptide amphiphile (PA) molecules that self-assemble and display the laminin epitope IKVAV at high density improved functional recovery after spinal cord injury (SCI) in two different species, rat and mouse, and in two different injury models, contusion and compression. The improvement required the IKVAV epitope and was not observed with the injection of an amphiphile displaying a nonbioactive sequence. To explore the mechanisms underlying these improvements, the number of serotonergic fibers in the lesioned spinal cord was compared in animals receiving the IKVAV-PA, a nonbioactive PA (PA control), or sham injection. Serotonergic fibers were distributed equally in all three groups rostral to the injury but showed a significantly higher density caudal to the injury site in the IKVAV PA-injected group. Furthermore, this difference was not present in the subacute phase following injury but appeared in the chronically injured cord. The IKVAV PA-injected groups also trended higher both in the total number neurons adjacent to the lesion and in the number of long propriospinal tract connections from the thoracic to the lumbar cord. IKVAV PA injection did not alter myelin thickness, total axon number caudal to the lesion, axon size distribution, or total axon area. Serotonin can promote stepping even in complete transection models, so the improved function produced by the IKVAV PA treatment may reflect the increased serotonergic innervation caudal to the lesion in addition to the previously demonstrated regeneration of motor and sensory axons through the lesion.

A case report of Milnacipran in phantom-limb pain

Introduction Phantom-limb sensations are so commonly reported after an amputation that they are considered normal. Up to 10% of amputees might report severe pain leading to a significant occupational dysfunction. And it is often overlooked because of limited breakthrough in understanding of aetiology and limited treatment options. I present a case report of PLP showing good response to Milnacipran – a novel antidepressant (SNRI) with dual mechanism of action through serotonin and norepinephrine reuptake inhibition similar to TCAs. Discussion In this case, patient had phantom-limb sensations along with phantom-limb pain which responded fairly to Tramadol but showed good response to Milnacipran. Milnacipran has already been approved by FDA for Fibromyalgia due to its action on noradrenergic and serotonergic fibres in descending inhibitory pain pathways as noted by Barkin and Fawcett (2000). Milnacipran is a selective serotonin and norepinephrine inhibitor (SNRI), sharing the class with other drugs likes Duloxetine and Venlafaxine. Patients on TCAs may not tolerate high doses due to their wide range of side effects like sedation, dry mouth, and constipation; thus limiting their use in PLP. Unlike TCAs, it does not act much on cholinergic or histaminic receptors, thus has minimal side effects. Thus, a molecule like Milnacipran, acting selectively on serotonin and norepinephrine receptors and having an efficacy profile similar to TCAs but without adverse effects, may show promising results in PLP.

Lack of analgesic efficacy of spinal ondansetron on thermal and mechanical hypersensitivity following spinal nerve ligation in the rat

The balance between descending inhibition and facilitation is thought to be disturbed in chronic pain states. Increased facilitation by spinally released serotonin has been suggested by demonstration that mechanically evoked neuronal responses of wide dynamic range neurons are inhibited by 5-HT3 receptor antagonists in rats following spinal nerve ligation (SNL) but not sham operation. Despite these physiologic data, the effects of spinal 5-HT3 receptor blockade on behavioral hypersensitivity and neurochemical alterations in spinal serotonergic system have not been thoroughly investigated following spinal nerve ligation in the rat. To test this, we acutely injected intrathecal ondansetron in rats between 14 and 30 days after SNL and assessed effects on thermal and mechanical hypersensitivity. We also determined the density of serotonergic nerve fibers, serotonin content and the levels of 5-HT3 receptors within the spinal cord at this time point. Intrathecal ondansetron (1, 3, 10, 30, and 100 μg) produced no effect on behavioral measures of thermal or mechanical hypersensitivity whereas intrathecal morphine (1 μg) and gabapentin (200 μg) partially reversed thermal and mechanical hypersensitivity following SNL. In addition, SNL did not alter the density of serotonergic fibers or 5-HT3 receptor immunoreactivity or spinal tissue content of 5-HT within the dorsal horn. These results do not support anatomic plasticity of descending serotonergic pathways or tonic 5-HT3 receptor activity in maintaining hypersensitivity after nerve injury and in contrast to previous studies fail to demonstrate an anti-hypersensitivity effect of intrathecal injection of the 5-HT3 receptor antagonist ondansetron. Importantly, behavioral measures of mechanical hypersensitivity assess threshold responses whereas physiological studies of mechanically evoked neuronal responses involve application of suprathreshold stimuli. Thus, suprathreshold or more intense stimuli may be necessary to recruit descending serotonergic facilitatory drive required to observe the inhibitory effects of ondansetron on spinal neuronal excitability and behavioral hypersensitivity.

Neurotoxic Effects of MDMA (Ecstasy) on the Developing Rodent Brain

The incidence of methamphetamine abuse is particularly high in adolescents and is a common problem among women of childbearing age, leading to an increasing number of children with prenatal exposure. MDMA (3,4-methylenedioxymethamphetamine, ecstasy) is an amphetamine-like stimulant and is known to induce apoptotic damage to fine serotonergic fibers in the adult rat brain. Little is known about toxic effects of MDMA and potential underlying molecular mechanisms in the developing brain. Here, we investigated whether MDMA exposure during the period of rapid brain growth causes neurodegeneration in the developing rat brain. MDMA significantly enhanced neuronal death in the brains of 6-day-old rat pups at a dose of 60 mg/kg, but no significant toxicity was detected at the ages of 14 and 21 days. Brain regions mainly affected were the cortex, septum, thalamus, hypothalamus and the cornu ammonis 1 region. To explore possible molecular mechanisms involved in this neurodegenerative process, we investigated the impact of MDMA on the expression of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor. Neonatal exposure of 6-day-old rats to MDMA triggered a considerable increase in cortical BDNF and NT-3 levels. Moreover, P7 CD1/BDNF knockout mice were noticeably more sensitive to MDMA exposure as compared to their wild-type age-matched littermates. These data suggest that a single injection of MDMA causes neurodegeneration in the neonatal rat brain. The upregulation of BDNF and NT-3 expression may indicate an important compensatory mechanism leading to the survival of neuronal cells in the developing brain.

Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury

Various types of induced pluripotent stem (iPS) cells have been established by different methods, and each type exhibits different biological properties. Before iPS cell-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Here we show the directed neural differentiation of murine iPS cells and examine their therapeutic potential in a mouse spinal cord injury (SCI) model. "Safe" iPS-derived neurospheres, which had been pre-evaluated as nontumorigenic by their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse brain, produced electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro. Furthermore, when the safe iPS-derived neurospheres were transplanted into the spinal cord 9 d after contusive injury, they differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in remyelination and induced the axonal regrowth of host 5HT(+) serotonergic fibers, promoting locomotor function recovery. However, the transplantation of iPS-derived neurospheres pre-evaluated as "unsafe" showed robust teratoma formation and sudden locomotor functional loss after functional recovery in the SCI model. These findings suggest that pre-evaluated safe iPS clone-derived neural stem/progenitor cells may be a promising cell source for transplantation therapy for SCI.

Serotonin fibre sprouting and increase in serotonin transporter immunoreactivity in the CA1 area of hippocampus in a triple transgenic mouse model of Alzheimer’s disease

Alzheimer's disease (AD) is a neurodegenerative disease that deteriorates cognitive functions and associated brain regions such as the hippocampus, being the primary cause of dementia. Serotonin (5-HT) is widely present in the hippocampus, being an important neurotransmitter involved in learning and memory. Although recent evidence suggests alterations in 5-HT neurotransmission in AD, it is not clear how hippocampal 5-HT innervation is modified. Here, we studied hippocampal 5-HT innervation by analysing: (i) the expression, density and distribution of 5-HT transporter (SERT)-immunoreactive fibres; (ii) the specific morphological characteristics of serotonergic fibres and their relation to amyloid plaques; and (iii) the total number of 5-HT neurons within the raphe nuclei in triple transgenic mouse model of AD. We used quantitative light microscopy immunohistochemistry comparing transgenic and non-transgenic animals of different ages (3, 6, 9, 12 and 18 months). The transgenic animals showed a significant increase in SERT fibres in the hippocampus in a subfield-, strata- and age-specific manner. The increase in SERT fibres was specific to the CA1 stratum lacunosum-moleculare. An increase in SERT fibres in transgenic animals was observed at 3 months (by 61%) and at 18 months (by 74%). No changes, however, were found in the total number of raphe 5-HT neurons at any age. Our results indicate that triple transgenic mice display changes in the expression of SERT and increased SERT fibres sprouting, which may account for imbalanced serotonergic neurotransmission associated with (or linked to) AD cognitive impairment.

Serotonin release variations during recovery of motor function after a spinal cord injury in rats

Current literature suggests that serotonin (5-HT) release within the ventral horn of the spinal cord plays a role in motor function. We hypothesized that endogenous 5-HT release is involved in the recovery of motor function after spinal cord injury. To appreciate the functional parameters of regenerating serotonergic fibers, a microdialysis probe was stereotactically implanted in the ventral horn of subhemi-lesioned rats. Microdialysis in combination with HPLC was used to measure concentrations of 5-HT in the lumbar ventral horn during periods of rest (90 min), treadmill run (60 min) and postexercise rest (90 min) for a 1-month time period of recovery following the surgical lesion. Within the same period of time, 5-HT levels varied significantly. A significant (202%) increase was observed at day 18 postlesion relative to day 8, and a 16.4% decrease was observed at day 34 relative to day 18. Treadmill exercise challenge induced a 10% decrease of 5-HT release relative to rest at days 18 and 34. In conclusion, overtime treadmill locomotor recovery is parallel to amounts (rest basal levels) and patterns (exercise and postexercise levels) of 5-HT release suggesting that changes in serotonergic system occurred within the same time frame than locomotor recovery using treadmill challenge.

Elevated BDNF protein level in cortex but not in hippocampus of MDMA-treated Dark Agouti rats: A potential link to the long-term recovery of serotonergic axons

3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”) is a widely used recreational drug known to cause selective long-term serotonergic damage. In this study, we examined the pattern of BDNF protein expression 1 day, 3, 8, 12 and 24 weeks after a single 15 mg/kg i.p. dose of MDMA to adolescent Dark Agouti rats. In parallel, we measured either tryptophan-hydroxylase immunoreactive (TpH IR) axon density, or [ 3H]-paroxetine-binding in parietal cortex and hippocampus, two brain areas known to have different recovery capacity after MDMA, to test whether BDNF-levels were associated with the long-term recovery of serotonergic fibers after a neurotoxic dose of MDMA. Both TpH IR axon density and [ 3H]-paroxetine-binding were significantly decreased 3 weeks after the treatment in both brain areas but while normalization in both parameters was found in parietal cortex 24 weeks after treatment, significant decreases remained evident in the hippocampus. In the parietal cortex, a significant reduction in BDNF protein levels was found in the acute phase after treatment (1 day), which was followed by a robust increase 8 weeks later and a return to control levels by 12 weeks. In contrast, no significant alteration of BDNF protein level was found in the hippocampus at any time points. This absence of any significant increase in BDNF protein levels in the hippocampus, and the persistence in this region of decreases in TpH IR axon density and [ 3H]-paroxetine-binding, raises the possibility that BDNF has an important role in the long-term recovery of serotonergic axons after MDMA treatment.

The hematopoietic factor granulocyte‐colony stimulating factor improves outcome in experimental spinal cord injury

Granulocyte-colony stimulating factor (G-CSF) is a potent hematopoietic factor that drives differentiation of neutrophilic granulocytes. We have recently shown that G-CSF also acts as a neuronal growth factor, protects neurons in vitro and in vivo, and has regenerative potential in various neurological disease models. Spinal cord injury (SCI) following trauma or secondary to skeletal instability is a terrible condition with no effective therapies available at present. In this study, we show that the G-CSF receptor is up-regulated upon experimental SCI and that G-CSF improves functional outcome in a partial dissection model of SCI. G-CSF significantly decreases apoptosis in an experimental partial spinal transsection model in the mouse and increases expression of the anti-apoptotic G-CSF target gene Bcl-X(L). In vitro, G-CSF enhances neurite outgrowth and branching capacity of hippocampal neurons. In vivo, G-CSF treatment results in improved functional connectivity of the injured spinal cord as measured by Mn(2+)-enhanced MRI. G-CSF also increased length of the dorsal corticospinal tract and density of serotonergic fibers cranial to the lesion center. Mice treated systemically with G-CSF as well as transgenic mice over-expressing G-CSF in the CNS exhibit a strong improvement in functional outcome as measured by the BBB score and gridwalk analysis. We show that G-CSF improves outcome after experimental SCI by counteracting apoptosis, and enhancing connectivity in the injured spinal cord. We conclude that G-CSF constitutes a promising and feasible new therapy option for SCI.

Pattern of distribution of serotonergic fibers to the thalamus of the rat

It is well established that serotonergic (5-hydroxytryptamine, 5-HT) fibers, mainly originating from the dorsal and median raphe nuclei of the brainstem, distribute throughout the forebrain, most heavily to 'limbic' forebrain structures. Few reports have examined the distribution of 5-HT fibers to the thalamus and none to our knowledge using immunoprocedures for the detection of the serotonin transporter (SERT)-a very sensitive marker for 5-HT fibers. Using immunohistochemical methods for SERT, we examined the pattern of distribution of 5-HT fibers to the thalamus in the rat. We show that serotonergic fibers are heavily concentrated in midline, intralaminar and association nuclei of the thalamus, and with the exception of the lateral geniculate complex, weakly distributed to principal nuclei of thalamus. Specifically, we demonstrate that 5-HT fibers are densely concentrated in the anteroventral, anteromedial and interanteromedial nuclei of the anterior thalamus, the paraventricular, rhomboid and reuniens nuclei of the midline thalamus, the central medial and central lateral nuclei of the intralaminar thalamus, the intermediodorsal nucleus, the lateral dorsal nucleus, and the dorsal and ventral lateral geniculate nuclei and intergeniculate leaflet of the LGN complex. Less densely innervated sites include the mediodorsal, paracentral, parafascicular, lateral posterior and submedial nuclei of thalamus. Remaining regions of the thalamus, largely consisting of principal nuclei, contained few 5-HT fibers. This pattern of 5-HT innervation indicates that serotonin/ serotonergic fibers mainly affect thalamic nuclei with connections to 'non-principal' or limbic regions of the cortex (or forebrain). This suggests that serotonergic fibers to the thalamus may exert a significant influence on affective and cognitive functions, possibly complementing the actions of 5-HT fibers to other parts of the brain involved in emotional and cognitive behaviors.

Serotonergic dystrophy induced by excess serotonin

Administration of certain serotonin-releasing amphetamine derivatives (fenfluramine and/or 3,4-methylenedioxymethamphetamine, MDMA, ‘ecstasy’) results in dystrophic serotonergic morphology in the mammalian brain. In addition to drug administration, dystrophic serotonergic neurites are also associated with neurodegenerative disorders. We demonstrate here that endogenously elevated serotonin in the Drosophila CNS induces aberrant enlarged varicosities, or spheroids, that are morphologically similar to dystrophic mammalian serotonergic fibers. In Drosophila these spheroids are specific to serotonergic neurons, distinct from typical varicosities, and form only after prolonged increases in cytoplasmic serotonin. Our results also suggest that serotonin levels during early development determine later sensitivity of spheroid formation to manipulations of the serotonin transporter (SERT). Elevated serotonin also interacts with canonical protein aggregation and autophagic pathways to form spheroids. The data presented here support a model in which excess cytoplasmic neurotransmitter triggers a cell-specific pathway inducing aberrant morphology in fly serotonergic neurons that may be shared in certain mammalian pathologies.

Novel combination strategies to repair the injured mammalian spinal cord

Due to the varied and numerous changes in spinal cord tissue following injury, successful treatment for repair may involve strategies combining neuroprotection (pharmacological prevention of some of the damaging intracellular cascades that lead to secondary tissue loss), axonal regeneration promotion (cell transplantation, genetic engineering to increase growth factors, neutralization of inhibitory factors, reduction in scar formation), and rehabilitation. Our goal has been to find effective combination strategies to improve outcome after injury to the adult rat thoracic spinal cord. Combination interventions tested have been implantation of Schwann cells (SCs) plus neuroprotective agents and growth factors administered in various ways, olfactory ensheathing cell (OEC) implantation, chondroitinase addition, or elevation of cyclic AMP. The most efficacious strategy in our hands for the acute complete transection/SC bridge model, including improvement in locomotion [Basso, Beattie, Bresnahan Scale (BBB)], is the combination of SCs, OECs, and chondroitinase administration (BBB 2.1 vs 6.6, 3 times more myelinated axons in the SC bridge, increased serotonergic axons in the bridge and beyond, and significant correlation between the number of bridge myelinated axons and functional improvement). We found the most successful combination strategy for a subacute spinal cord contusion injury (12.5–mm, 10–g weight, MASCIS impactor) to be SCs and elevation of cyclic AMP (BBB 10.4 vs 15, significant increases in white matter sparing, in myelinated axons in the implant, and in responding reticular formation and red and raphe nuclei, and a significant correlation between the number of serotonergic fibers and improvement in locomotion). Thus, in two injury paradigms, these combination strategies as well as others studied in our laboratory have been found to be more effective than SCs alone and suggest ways in which clinical application may be developed.

VGLUT3 (Vesicular Glutamate Transporter Type 3) Contribution to the Regulation of Serotonergic Transmission and Anxiety

Three different subtypes of H(+)-dependent carriers (named VGLUT1-3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT(1A)-mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3- and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.

Differential effects of neonatal hypoxic–ischemic brain injury on brainstem serotonergic raphe nuclei

Serotonergic fibres have a pervasive innervation of hypoxic–ischemic (HI)-affected areas in the neonatal brain and serotonin (5-HT) is pivotal in numerous neurobehaviours that match many HI-induced deficits. However, little is known about how neonatal HI affects the serotonergic system. We therefore examined whether neonatal HI can alter numbers of serotonergic raphe neurons in specific sub-divisions of the midbrain and brainstem since these nuclei are the primary sources of serotonin throughout the central nervous system (CNS). We utilised an established neonatal HI model in the postnatal day 3 (P3) rat pup (right common carotid artery ligation + 30 min 6% O 2) and determined the effects of P3 HI on 5-HT counts in 5 raphe sub-divisions in the midbrain and brainstem one and six weeks later. After P3 HI, numbers of 5-HT-positive neurons were significantly decreased in the dorsal raphe dorsal, dorsal raphe ventrolateral and dorsal raphe caudal nuclei on P10 but only in the dorsal raphe dorsal and dorsal raphe ventrolateral nuclei on P45. In contrast, P3 HI did not alter counts in the dorsal raphe interfascicular and raphe magnus nuclei. We also discovered that P3 HI significantly reduces brainstem SERT protein expression; the key regulator of 5-HT in the CNS. In conclusion, neonatal HI injury caused significant disruption of the brainstem serotonergic system that can persist for up to six weeks after the insult. The different vulnerabilities of serotonergic populations in specific raphe nuclei suggest that certain raphe nuclei may underpin neurological deficits in HI-affected neonates through to adulthood.