Cellular correlates of long-term sensitization in Aplysia

Although in vitro analyses of long-term changes in the sensorimotor connection of Aplysia have been used extensively to understand long-term sensitization, relatively little is known about the ways in which the connection is modified by learning in vivo. Moreover, sites other than the sensory neurons might be modified as well. In this paper, several different biophysical properties of sensory neurons, motor neurons, and LPl17, an identified interneuron, were examined. Membrane properties of sensory neurons, which were expressed as increased excitability and increased spike afterdepolarization, were affected by the training. The biophysical properties of motor neurons also were affected by training, resulting in hyperpolarization of the resting membrane potential and a decrease in spike threshold. These results suggest that motor neurons are potential loci for storage of the memory in sensitization. The strength of the connection between sensory and motor neurons was affected by the training, although the connection between LPl17 and the motor neuron was unaffected. Biophysical properties of LPl17 were unaffected by training. The results emphasize the importance of plasticity at sensory-motor synapses and are consistent with the idea that there are multiple sites of plasticity distributed throughout the nervous system.     

Mode-switching of a voltage-gated cation channel is mediated by a protein kinase A-regulated tyrosine phosphatase

Tyrosine kinases and tyrosine phosphatases are abundant in central nervous system tissue, yet the role of these enzymes in the modulation of neuronal excitability is unknown. Patch-clamp studies of an Aplysia voltage-gated cation channel now demonstrate that a tyrosine phosphatase endogenous to excised patches determines both the gating mode of the channel and the response of the channel to protein kinase A. Moreover, a switch in gating modes similar to that triggered by the phosphatase occurs at the onset of a prolonged change in the excitability of Aplysia bag cell neurons.     

Insulin receptor in Aplysia neurons: characterization, molecular cloning, and modulation of ion currents

We have isolated the cDNA for a tyrosine kinase receptor that is expressed in the nervous system of Aplysia californica and that is similar to the vertebrate insulin receptor. Binding studies and immunocytochemical staining show that the receptor is abundant in the bag cell neurons. Application of vertebrate insulin to clusters of bag cell neurons stimulates the phosphorylation of the receptor on tyrosine residues, and exposure of isolated bag cell neurons to insulin produces an increase in height and a decrease in duration of the action potentials that can be detected within 15-30 min. These effects were not seen with insulin-like growth factor-1. In voltage-clamped neurons, insulin produces an increase in the amplitude of the voltage-dependent Ca2+ current that can be blocked by preincubation with herbimycin A, an inhibitor of tyrosine kinases. Insulin also enhances a delayed K+ current. We suggest that insulin-like peptides regulate the excitability of the bag cell neurons.     

Neuropeptides in the skin: interactions between the neuroendocrine and the skin immune systems

The interaction between components of the nervous system and multiple target cells in the cutaneous immune system has been receiving increasing attention. It has been observed that certain skin diseases such as psoriasis and atopic dermatitis have a neurogenic component. Neuropeptides released by sensory nerves that innervate the skin and often contact epidermal and dermal cells can directly modulate functions of keratinocytes, Langerhans cells (LC), mast cells, dermal microvascular endothelial cells and infiltrating immune cells. Among these neuropeptides the tachykinins substance P (SP) and neurokinin A (NKA), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and somatostatin (SOM) have been reported to effectively modulate skin and immune cell functions such as cell proliferation, cytokine production or antigen presentation under physiological or pathophysiological conditions. Expression and regulation of their corresponding receptors that are expressed on a variety of skin cells as well as the presence of neuropeptide-specific peptidases such as neutral endopeptidase (NEP) or angiotensin-converting enzyme (ACE) determine the final biological response mediated by these peptides on the target cell or tissue. Likewise, skin cells like keratinocytes or fibroblasts are a source for neurotrophins such as nerve growth factor that are required not only for survival and regeneration of sensory neurons but also to control responsiveness of these neurons to external stimuli. Therefore, neuropeptides, neuropeptide receptors, neuropeptide-degrading enzymes and neurotrophins participate in a complex, interdependent network of mediators that modulate skin inflammation, wound healing and the skin immune system. This review will focus on recent studies demonstrating the role of tachykinins, CGRP, SOM and VIP and their receptors and neuropeptide-degrading enzymes in mediating neurogenic inflammation in the skin.     

A new class of noninactivating K+ channels from aplysia capable of contributing to the resting potential and firing patterns of neurons

From the nervous system of Aplysia, we have cloned a new class of noninactivating K+ channels (aKv5.1) that are activated at low voltage and are capable of contributing to the resting potential and firing patterns of neurons. Expression of aKv5.1 in Aplysia neuron R15 revealed that aKv5.1 exerts an unusual control over cell excitability; it increased the resting potential by more than 20 mV and abolished the spontaneous bursting activity of the cell. In its ability to suppress the endogenous rhythm of R15, aKv5.1 differs in its actions from transient, inactivating K+ channels such as aKv1.1a, an Aplysia homolog of Shaker. aKv1.1a shortens the duration of the spike and increases the afterpotential, but does not suppress bursting. Thus, by expressing different classes of K+ channels, it is possible to redesign, in specific ways, the signaling capabilities of specific, identified neurons.     

Stem cells in the central nervous system

In the vertebrate central nervous system, multipotential cells have been identified in vitro and in vivo. Defined mitogens cause the proliferation of multipotential cells in vitro, the magnitude of which is sufficient to account for the number of cells in the brain. Factors that control the differentiation of fetal stem cells to neurons and glia have been defined in vitro, and multipotential cells with similar signaling logic can be cultured from the adult central nervous system. Transplanting cells to new sites emphasizes that neuroepithelial cells have the potential to integrate into many brain regions. These results focus attention on how information in external stimuli is translated into the number and types of differentiated cells in the brain. The development of therapies for the reconstruction of the diseased or injured brain will be guided by our understanding of the origin and stability of cell type in the central nervous system.     

Developmental dissociation of excitability and secretory ability in Aplysia bag cell neurons

1. Despite the considerable progress made in understanding the role of electrical activity in triggering secretion, the developmental relationships between excitability and secretion are not well understood. The well-characterized bag cell neurons of Aplysia provide an advantageous system in which to investigate developmental interactions of these two key properties of neurons. 2. A prolonged afterdischarge triggers egg laying hormone (ELH) secretion in mature bag cell neurons. To investigate secretion in the developmental framework of excitability, we first examined whether immature neurons, which are incapable of the mature form of excitability (afterdischarge), contain ELH and whether this hormone is packaged in vesicles. We used immunoelectron microscopy to compare vesicular localization of ELH and to compare the size and density of ELH-containing vesicles in neurons from adult and juvenile Aplysia. This comparison revealed that immature neurons contain ELH in vesicles in the size range of secretory vesicles. However, they lack a class of large vesicles (> 250 nm in diameter) that is characteristic of mature neurons. 3. To investigate whether the ELH contained in immature bag cell neurons could be secreted in response to electrical activity, we used the potassium channel blocker tetraethylammonium (TEA) combined with nerve stimulation to depolarize neurons from both juvenile animals (ovotestes do not contain eggs) and from adult Aplysia (ovotestes contain eggs). Using radioimmunoassay, we have found that the duration and amount of ELH secreted from bag cell neurons from juvenile Aplysia in response to TEA does not depend on whether or not the cells can be induced to afterdischarge, and the amount and duration of ELH secreted from bag cell neurons of juvenile Aplysia (whether or not they afterdischarged) differed from those secreted by adult neurons. However, by normalizing for body size, we found that the final estimated hemolymph concentration of ELH would be similar in juvenile and adult animals. 4. We investigated the potential functional significance of secretion of bag cell hormones in juvenile Aplysia by attempting to bypass the bag cell neurons and directly activate downstream elements with extract from adult bag cell neurons (BCE), known to contain ELH and other peptides. We found that juvenile Aplysia exhibit at least one component of egg-laying behavior, cessation of locomotion, in response to BCE during a developmental period (as measured by weight) in which they normally would possess neurons incapable of afterdischarge. Thus developmental regulation of excitability in the bag cell neurons may prevent inappropriate hormone release and subsequent premature expression of reproductive behaviors.     

Activation of protein kinase A contributes to the expression but not the induction of long-term hyperexcitability caused by axotomy of Aplysia sensory neurons

Nociceptive sensory neurons (SNs) in Aplysia provide useful models to study both memory and adaptive responses to nerve injury. Induction of long-term memory in many species, including Aplysia, is thought to depend on activation of cAMP-dependent protein kinase (PKA). Because Aplysia SNs display similar alterations in models of memory and after nerve injury, a plausible hypothesis is that axotomy triggers memory-like modifications by activating PKA in damaged axons. The present study disproves this hypothesis. SN axotomy was produced by (1) dissociation of somata from the ganglion [which is shown to induce long-term hyperexcitability (LTH)], (2) transection of neurites of dissociated SNs growing in vitro, or (3) peripheral nerve crush. Application of the competitive PKA inhibitor Rp-8-CPT-cAMPS at the time of axotomy failed to alter the induction of LTH by each form of axotomy, although the inhibitor antagonized hyperexcitability produced by 5-HT application. Strong activation of PKA in the nerve by coapplication of a membrane-permeant analog of cAMP and a phosphodiesterase inhibitor was not sufficient to induce LTH of either the SN somata or axons. Furthermore, nerve crush failed to activate axonal PKA or stimulate its retrograde transport. Therefore, PKA activation plays little if any role in the induction of LTH by axotomy. However, the expression of LTH was reduced by intracellular injection of the highly specific PKA inhibitor PKI several days after nerve crush. This suggests that long-lasting activation of PKA in or near the soma contributes to the maintenance of long-term modifications produced by nerve injury.     

ADP-ribosylation of serum proteins: evaluation as a potential tumor marker

The low-affinity p75 receptor for nerve growth factor (p75NGFR) has been implicated in mediating neuronal cell death in vitro. A recent in vitro study from our laboratory showed that the death of sensory neurons can be prevented by reducing the levels of p75NGFR with antisense oligonucleotides. To determine if p75NGFR also functions as a death signal in vivo, we have attempted to reduce its expression in peripheral sensory neurons by applying antisense oligonucleotides to the proximal end of the transected sciatic or median and ulnar nerves. We report here that antisense oligonucleotides, when applied to the proximal stump of a transected peripheral nerve, are retrogradely transported and effectively reduce p75NGFR protein levels in sensory neurons located in the dorsal root ganglia. Furthermore, treatment of the proximal nerve stump with antisense p75NGFR oligonucleotides significantly reduced the loss of these axotomized sensory neurons. These findings further support the view that p75NGFR is a death signaling molecule and that it signals death in axotomized neurons in the neonatal sensory nervous system.     

Rebound insomnia after hypnotic withdrawal in insomniac outpatients

Interleukin-2 (IL-2) is not only an important immunoregulatory molecule: it is also an important neuroregulatory molecule in the CNS. Investigations in vivo and in vitro have elucidated the following: IL-2 and IL-2 receptors (IL-2R) have been detected in the brain; IL-2 promotes survival and neurite extension of cultured neurons, stimulates oligodendrocyte proliferation and maturation, affects the hypothalamic-pituitary function and produces behavioral and electrocorticogram spectrum changes. Investigations in some laboratories, including ours, have also demonstrated an analgesic effect of IL-2 in the CNS. The molecular structure of the analgesic domain of IL-2 is distinct from the immune domain mediating immunoregulatory effects. A possible mechanism of action of cytokines on the CNS and the immune system or both exists: multiple actions of cytokines could be mediated by distinct domains or functional sites of cytokines interacting with different receptors or receptor subtypes.     

List-mode likelihood: EM algorithm and image quality estimation demonstrated on 2-D PET

Interleukin-6 (IL-6) is a multifunctional cytokine whose actions include modulation of proliferation, differentiation, and maturation of hemapoietic progenitors and other cell lineages; growth regulation of certain carcinoma cell lines; and control of cellular metabolic activities. Initially described in terms of its activities in the immune system and inflammation, accumulating evidence supports an essential role of IL-6 in the development, differentiation, regeneration and degeneration of neurons in the peripheral and central nervous system. We have previously demonstrated that immunoreactive-like IL-6 protein is significantly elevated in the spinal cord in response to peripheral nerve injury that results in neuropathic pain behaviors in the rat. In the current study, our objective was to determine if the source of IL-6 protein was endogenous to the central nervous system by measuring any detectable increases in spinal IL-6 mRNA expression following established mononeuropathy procedures associated with neuropathic pain: spinal nerve cryoneurolysis (SPCN) or spinal nerve tight ligation (SPTL). Using in situ hybridization and a digoxigenin-labeled oligonucleotide, IL-6 mRNA in neurons was significantly elevated at 3 and 7 days post SPCN and 7 days post SPTL in both dorsal and ventral horns. The cellular localization of the IL-6 mRNA expression was predominately neuronal as confirmed by NeuN serial staining. For example, in the SPCN 7 day group, IL-6 mRNA cell profiles in the ipsilateral dorsal horn were significantly different from the normal group (38.7+/-12.8 vs. 4.89+/-1.6, p<0.001). These data demonstrate the central, spinal production of a proinflammatory cytokine in response to a peripheral nerve injury. In addition, these results add to the growing body of literature implicating these immune products, cytokines, as potential neuromodulators/neurotransmitters and provides further evidence for their role in the nociceptive processing which leads to chronic pain.     

Developmentally regulated expression of persyn, a member of the synuclein family, in skin

Synucleins constitute a group of unique, evolutionarily conserved proteins that are expressed predominantly in neurons of the central and peripheral nervous system. Although the normal cellular functions of synucleins are not clear, these proteins have been implicated in various neurodegenerative conditions in humans. We found that persyn, a recently characterized member of the synuclein family, is expressed not only in the nervous system but also in the stratum granulosum of the epidermis of neonatal and adult mice. This finding together with our recent observations that persyn influences neurofilament network integrity in sensory neurons raises the possibility that persyn in skin could be involved in modulation of the keratin network.     

Identification of an 8-lipoxygenase pathway in nervous tissue of Aplysia californica

Arachidonic acid is converted to (8R)-hydroperoxyeicosa-5,9,11, 14-tetraenoic acid (8-HPETE) during incubations with homogenates of the central nervous system of the marine mollusc, Aplysia californica. 8-HPETE can be reduced to the corresponding hydroxy acid or be enzymatically converted to a newly identified metabolite, 8-ketoeicosa-5,9,11,14-tetraenoic acid (8-KETE). These metabolites were identified by high performance liquid chromatography, UV absorbance, and gas chromatography/mass spectrometry. Stereochemical analysis of the products demonstrate that the neuronal enzyme is an (8R)-lipoxygenase. Previously we have shown that the neurotransmitters, histamine and Phe-Met-Arg-Phe-amide, activate 12-lipoxygenase metabolism in isolated identified Aplysia neurons. We now show that acetylcholine activates the (8R)-lipoxygenase pathway within intact nerve cells. Thus, both (12S)- and (8R)-lipoxygenase co-exist in intact Aplysia nervous tissue but are differentially activated by several neurotransmitters. The precise physiological role of the 8-lipoxygenase products is currently under investigation, but by analogy to the well-described 12-lipoxygenase pathway, we suggest that (8R)-HPETE and 8-KETE may serve as second messengers in Aplysia cholinoceptive neurons.     

Long-term changes in excitability induced by protein kinase C activation in Aplysia sensory neurons

Protein kinases A (PKA) and C (PKC) play a central role as intracellular transducers during simple forms of learning in Aplysia. These two proteins seem to cooperate in mediating the different forms of plasticity underlying behavioral modifications of defensive reflexes in a state- and time-dependent manner. Although short- and long-term changes in the synaptic efficacy of the connections between mechanosensory neurons and motoneurons of the reflex have been well characterized, there is also a distinct intermediate phase of plasticity that is not as well understood. Biochemical and physiological experiments have suggested a role for PKC in the induction and expression of this form of facilitation. In this report, we demonstrate that PKC activation can induce both intermediate- and long-term changes in the excitability of sensory neurons (SNs). Short application of 4beta-phorbol ester 12,13-dibutyrate (PDBU), a potent activator of PKC, produced a long-lasting increase in the number of spikes fired by SNs in response to depolarizing current pulses. This effect was observed in isolated cell culture and in the intact ganglion; it was blocked by a selective PKC inhibitor (chelerythrine). Interestingly, the increase in excitability measured at an intermediate-term time point (3 h) after treatment was independent of protein synthesis, while it was disrupted at the long-term (24 h) time point by the general protein synthesis inhibitor, anisomycin. In addition to suggesting that PKC as well as PKA are involved in long-lasting excitability changes, these findings support the idea that memory formation involves multiple stages that are mechanistically distinct at the biochemical level.     

Coroners'' records of rural and non-rural cases of youth suicide in New South Wales

The cytokine interleukin-1 beta (IL-1beta) is an evolutionarily conserved molecule that was originally identified in the immune system. In addition to regulating peripheral immune responses, IL-1beta plays an important role in mediating neural-immune interactions and regulating glial activities during healing and repair in the damaged nervous system. Active IL-1beta is produced by interleukin-converting enzyme (ICE), a caspase thought to be involved in the induction of apoptosis. We report that, in the developing frog, Xenopus laevis, IL-1beta and the IL-1 type 1 receptor proteins are coexpressed in specific neurons that comprise early sensory-motor circuits. IL-1beta and IL-1 type 1 receptor proteins are colocalized in specific midbrain and hindbrain reticular cells, including Mauthner's neuron; specific cells in the trigeminal (fifth), lateral line (seventh), and vestibular (eighth) cranial ganglia; oculomotor neurons; and the primordial Purkinje cells of the lateral cerebellar auricle. In the spinal cord, Rohon-Beard sensory neurons, dorsal root ganglion cells, and primary motoneurons are immunopositive. Anteriorly, the olfactory pits, olfactory nerves, and olfactory bulbs are labeled, as are retinal cells, especially photoreceptor inner segments. With regard to the function of IL-1beta during neural development, IL-1beta and its type 1 receptor are present throughout the course of neural development in identifiable, long-lived neurons, such as Mauthner's neuron. These and other data suggest that IL-1beta and its type 1 receptor may be involved in the maintenance of cell survival rather than induction of neuronal death.     

Mutation in the phosphorylation sites of MAP kinase blocks learning-related internalization of apCAM in Aplysia sensory neurons

The synaptic growth that accompanies 5-HT-induced long-term facilitation of the sensory to motor neuron connection in Aplysia is associated with the internalization of apCAM at the surface membrane of the sensory neuron. We have now used epitope tags to examine the fate of each of the two apCAM isoforms (membrane bound and GPI-linked) and find that only the transmembrane form is internalized. This internalization can be blocked by overexpression of transmembrane constructs with a single point mutation in the two MAPK consensus sites, as well as by injection of a specific MAPK antagonist into sensory neurons. These data suggest MAPK phosphorylation at the membrane is important for the internalization of apCAMs and, thus, may represent an early regulatory step in the growth of new synaptic connections that accompanies long-term facilitation.     

Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and cytokines

Reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) was induced by UV irradiation of the corneas of latently infected mice. Immunocytochemistry was used to monitor the dynamics of cytokine (interleukin-2 [IL-2], IL-4, IL-6, IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and viral antigen production in the TG and the adjacent central nervous system on days 1 to 4, 6, 7, and 10 after irradiation. UV irradiation induced increased expression of IL-6 and TNF-alpha from satellite cells in uninfected TG. In latently infected TG, prior to reactivation, all satellite cells were TNF-alpha+ and most were also IL-6(+). Reactivation, evidenced by HSV-1 antigens and/or infiltrating immune cells, occurred in 28 of 45 (62%) TG samples. Viral antigens were present in the TG in neurons, often disintegrating on days 2 to 6 after irradiation. Infected neurons were usually surrounded by satellite cells and the foci of immune cells producing TNF-alpha and/or IL-6. IL-4(+) cells were detected as early as day 3 and were more numerous by day 10 (a very few IL-2(+) and/or IFN-gamma+ cells were seen at this time). No IL-10 was detected at any time. Our observations indicate that UV irradiation of the cornea may modulate cytokine production by satellite cells. We confirm that neurons are the site of reactivation and that they probably do not survive this event. The predominance of TNF-alpha and IL-6 following reactivation parallels primary infection in the TG and suggests a role in viral clearance. The presence of Th2-type cytokines (IL-4 and IL-6) indicates a role for antibody. Thus, several clearance mechanisms may be at work.