Peripheral Nerve Ligation Research Articles

Peripheral Nerve Ligation Elicits Widespread Alterations in Cortical Sensory Evoked and Spontaneous Activity

Peripheral neuropathies result in adaptation in primary sensory and other regions of cortex, and provide a framework for understanding the localized and widespread adaptations that arise from altered sensation. Mesoscale cortical imaging achieves high temporal resolution of activity using optical sensors of neuronal activity to simultaneously image across a wide expanse of cortex and capture this adaptation using sensory-evoked and spontaneous cortical activity. Saphenous nerve ligation in mouse is an animal model of peripheral neuropathy that produces hyperalgesia circumscribed to the hindlimb. We performed saphenous nerve ligation or sham, followed by mesoscale cortical imaging using voltage sensitive dye (VSD) after ten days. We utilized subcutaneous electrical stimulation at multiple stimulus intensities to characterize sensory responses after ligation or sham, and acquired spontaneous activity to characterize functional connectivity and large scale cortical network reorganization. Relative to sham animals, the primary sensory-evoked response to hindlimb stimulation in ligated animals was unaffected in magnitude at all stimulus intensities. However, we observed a diminished propagating wave of cortical activity at lower stimulus intensities in ligated animals after hindlimb, but not forelimb, sensory stimulation. We simultaneously observed a widespread decrease in cortical functional connectivity, where midline association regions appeared most affected. These results are consistent with localized and broad alterations in intracortical connections in response to a peripheral insult, with implications for novel circuit level understanding and intervention for peripheral neuropathies and other conditions affecting sensation.

Peripheral nerve injury-induced rearrangement of neural circuit in the spinal dorsal horn revealed by cross-correlation analysis.

Peripheral nerve injury often induces abnormal pain states, such as hyperalgesia and allodynia. In this study, we attempted to elucidate how neurons are synaptically integrated into the neuronal circuitry in the spinal dorsal horn and how synaptic connectivity patterns among dorsal horn neurons are altered by peripheral nerve injury. Experiments were performed on 6-8-week-old ICR mice. Partial sciatic nerve ligation was performed. Transverse slices of the lumbar spinal cord were prepared. Spike activities were simultaneously recorded from multiple neurons in the superficial dorsal horn (SDH) using a multi-electrode array system, and cross-correlograms between spike trains of neuron pairs were constructed. In sham-operated control mice, except for the flat cross-correlogram, the most common pattern was a cross-correlogram suggestive of common excitatory synaptic inputs to neuronal pairs. Peripheral nerve ligation increased the incidences of cross-correlograms suggestive of common excitatory synaptic inputs and excitatory synaptic connections, and decreased that of inhibitory synaptic connections. Additionally, bath-applied capsaicin, an agonist for transient receptor potential vanilloid 1 receptor, increased the frequency of action potentials. The effects of capsaicin stimulation on the incidence of cross-correlograms with various patterns were significantly different between sham-operated control and sciatic nerve-ligated mice. The present observations seem to indicate that neurons in the SDH form excitatory and/or inhibitory synapses with nearby neurons, and that synaptic connections among neurons in the SDH may remarkably change after the development of neuropathic pain.

An Improved Rodent Model of Trigeminal Neuropathic Pain by Unilateral Chronic Constriction Injury of Distal Infraorbital Nerve.

The orofacial pain caused by trigeminal nerve damage is severe and perhaps more debilitating than other types of neuropathic pain. However, studies on trigeminal neuropathic pain remain limited. This is largely because of the lack of proper animal models because of the complexity of the existing surgical procedures required to induce trigeminal nerve injury. Our improved dIoN-CCI model is likely to make it more accessible to study the cellular and molecular mechanisms of neuropathic pain caused by trigeminal nerve damage.

Plasticity of DNA methylation in a nerve injury model of pain

The response of the peripheral nervous system (PNS) to injury may go together with alterations in epigenetics, a conjecture that has not been subjected to a comprehensive, genome-wide test. Using reduced representation bisulfite sequencing, we report widespread remodeling of DNA methylation in the rat dorsal root ganglion (DRG) occurring within 24 h of peripheral nerve ligation, a neuropathy model of allodynia. Significant (P < 10−4) cytosine hyper- and hypo-methylation was found at thousands of CpG sites. Remodeling occurred outside of CpG islands. Changes affected genes with known roles in the PNS, yet methylome remodeling also involved genes that were not linked to neuroplasticity by prior evidence. Consistent with emerging models relying on genome-wide methylation and RNA-seq analysis of promoter regions and gene bodies, variation of methylation was not tightly linked with variation of gene expression. Furthermore, approximately 44% of the dynamically changed CpGs were located outside of genes. We compared their positions with the intergenic, tissue-specific differentially methylated CpGs (tDMCs) of an independent experimental set consisting of liver, spleen, L4 control DRG, and muscle. Dynamic changes affected those intergenic CpGs that were different between tissues (P < 10−15) and almost never the invariant portion of the methylome (those CpGs that were identical across all tissues). Our findings—obtained in mixed tissue—show that peripheral nerve injury leads to methylome remodeling in the DRG. Future studies may address which of the cell types found in the DRG, such as specific groups of neurons or non-neuronal cells are affected by which aspect of the observed methylome remodeling.

High-voltage-activated calcium current subtypes in mouse DRG neurons adapt in a subpopulation-specific manner after nerve injury.

Changes in ion channel function and expression are characteristic of neuropathic pain. Voltage-gated calcium channels (VGCCs) are integral for neurotransmission and membrane excitability, but relatively little is known about changes in their expression after nerve injury. In this study, we investigate whether peripheral nerve ligation is followed by changes in the density and proportion of high-voltage-activated (HVA) VGCC current subtypes in dorsal root ganglion (DRG) neurons, the contribution of presynaptic N-type calcium channels in evoked excitatory postsynaptic currents (EPSCs) recorded from dorsal horn neurons in the spinal cord, and the changes in expression of mRNA encoding VGCC subunits in DRG neurons. Using C57BL/6 mice [8- to 11-wk-old males (n = 91)] for partial sciatic nerve ligation or sham surgery, we performed whole cell patch-clamp recordings on isolated DRG neurons and dorsal horn neurons and measured the expression of all VGCC subunits with RT-PCR in DRG neurons. After nerve injury, the density of P/Q-type current was reduced overall in DRG neurons. There was an increase in the percentage of N-type and a decrease in that of P/Q-type current in medium- to large-diameter neurons. No changes were found in the contribution of presynaptic N-type calcium channels in evoked EPSCs recorded from dorsal horn neurons. The α2δ-1 subunit was upregulated by 1.7-fold and γ-3, γ-2, and β-4 subunits were all downregulated 1.7-fold in injured neurons compared with sham-operated neurons. This comprehensive characterization of HVA VGCC subtypes in mouse DRG neurons after nerve injury revealed changes in N- and P/Q-type current proportions only in medium- to large-diameter neurons.

GluA1 phosphorylation contributes to postsynaptic amplification of neuropathic pain in the insular cortex.

Long-term potentiation of glutamatergic transmission has been observed after physiological learning or pathological injuries in different brain regions, including the spinal cord, hippocampus, amygdala, and cortices. The insular cortex is a key cortical region that plays important roles in aversive learning and neuropathic pain. However, little is known about whether excitatory transmission in the insular cortex undergoes plastic changes after peripheral nerve injury. Here, we found that peripheral nerve ligation triggered the enhancement of AMPA receptor (AMPAR)-mediated excitatory synaptic transmission in the insular cortex. The synaptic GluA1 subunit of AMPAR, but not the GluA2/3 subunit, was increased after nerve ligation. Genetic knock-in mice lacking phosphorylation of the Ser845 site, but not that of the Ser831 site, blocked the enhancement of the synaptic GluA1 subunit, indicating that GluA1 phosphorylation at the Ser845 site by protein kinase A (PKA) was critical for this upregulation after nerve injury. Furthermore, A-kinase anchoring protein 79/150 (AKAP79/150) and PKA were translocated to the synapses after nerve injury. Genetic deletion of adenylyl cyclase subtype 1 (AC1) prevented the translocation of AKAP79/150 and PKA, as well as the upregulation of synaptic GluA1-containing AMPARs. Pharmacological inhibition of calcium-permeable AMPAR function in the insular cortex reduced behavioral sensitization caused by nerve injury. Our results suggest that the expression of AMPARs is enhanced in the insular cortex after nerve injury by a pathway involving AC1, AKAP79/150, and PKA, and such enhancement may at least in part contribute to behavioral sensitization together with other cortical regions, such as the anterior cingulate and the prefrontal cortices.

Smad‐interacting protein 1 affects acute and tonic, but not chronic pain

Smad-interacting protein 1 (also named Zeb2 and Zfhx1b) is a transcription factor that plays an important role in neuronal development and, when mutated, causes Mowat-Wilson syndrome (MWS). A corresponding mouse model carrying a heterozygous Zeb2 deletion was comprehensively analysed in the German Mouse Clinic. The most prominent phenotype was the reduced pain sensitivity. In this study, we investigated the role of Zeb2 in inflammatory and neuropathic pain. For this, we tested mutant Zeb2 animals in different models of inflammatory pain like abdominal constriction, formalin and carrageenan test. Furthermore, we studied the pain reactivity of the mice after peripheral nerve ligation. To examine the nociceptive transmission of primary sensory dorsal root ganglia (DRG) neurons, we determined the neuronal activity in the spinal dorsal horn after the formalin test using staining of c-Fos. Next, we characterized the neuronal cell population in the DRGs and in the sciatic nerve to study the effect of the Zeb2 mutation on peripheral nerve morphology. The present data show that Zeb2 is involved in the development of primary sensory DRG neurons, especially of C- and Aδ fibres. These alterations contribute to a hypoalgesic phenotype in inflammatory but not in neuropathic pain in these Zeb2(+/-) mice. Our data suggest that the under-reaction to pain observed in MWS patients results from a reduced responsivity to nociceptive stimulation rather than an inability to communicate discomfort.

HCN1 Channels as Targets for Anesthetic and Nonanesthetic Propofol Analogs in the Amelioration of Mechanical and Thermal Hyperalgesia in a Mouse Model of Neuropathic Pain

Chronic pain after peripheral nerve injury is associated with afferent hyperexcitability and upregulation of hyperpolarization-activated, cyclic nucleotide-regulated (HCN)-mediated IH pacemaker currents in sensory neurons. HCN channels thus constitute an attractive target for treating chronic pain. HCN channels are ubiquitously expressed; analgesics targeting HCN1-rich cells in the peripheral nervous system must spare the cardiac pacemaker current (carried mostly by HCN2 and HCN4) and the central nervous system (where all four isoforms are expressed). The alkylphenol general anesthetic propofol (2,6-di-iso-propylphenol) selectively inhibits HCN1 channels versus HCN2-HCN4 and exhibits a modest pharmacokinetic preference for the periphery. Consequently, we hypothesized that propofol, and congeners, should be antihyperalgesic. Alkyl-substituted propofol analogs have different rank-order potencies with respect to HCN1 inhibition, GABA(A) receptor (GABA(A)-R) potentiation, and general anesthesia. Thus, 2,6- and 2,4-di-tertbutylphenol (2,6- and 2,4-DTBP, respectively) are more potent HCN1 antagonists than propofol, whereas 2,6- and 2,4-di-sec-butylphenol (2,6- and 2,4-DSBP, respectively) are less potent. In contrast, DSBPs, but not DTBPs, enhance GABA(A)-R function and are general anesthetics. 2,6-DTBP retained propofol's selectivity for HCN1 over HCN2-HCN4. In a peripheral nerve ligation model of neuropathic pain, 2,6-DTBP and subhypnotic propofol are antihyperalgesic. The findings are consistent with these alkylphenols exerting analgesia via non-GABA(A)-R targets and suggest that antagonism of central HCN1 channels may be of limited importance to general anesthesia. Alkylphenols are hydrophobic, and thus potential modifiers of lipid bilayers, but their effects on HCN channels are due to direct drug-channel interactions because they have little bilayer-modifying effect at therapeutic concentrations. The alkylphenol antihyperalgesic target may be HCN1 channels in the damaged peripheral nervous system.

Differential expression of NMDA receptors in serotonergic and/or GABAergic neurons in the midbrain periaqueductal gray of the mouse

N-methyl-d-aspartate (NMDA) receptors expressed in the midbrain periaqueductal gray (PAG) exert various physiological functions. The PAG contains various neurotransmitter phenotypes, which include GABAergic neurons and serotonergic neurons. In the present experiments, we made tight-seal whole-cell recordings from GABAergic and/or serotonergic neurons in mouse PAG slices and analyzed NMDA and non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation. The NMDA/non-NMDA ratio of EPSC amplitude was high and the decay time course of NMDA-EPSC was slow in non-serotonergic/GABAergic neurons. In contrast, serotonergic neurons exhibited a low NMDA/non-NMDA ratio and a fast decay time course of NMDA-EPSC. Peripheral nerve ligation-induced chronic pain was associated with an increased NMDA/non-NMDA ratio in serotonergic neurons. Additionally, single-cell real-time RT-PCR analysis showed that peripheral nerve ligation up-regulated NR2B subunit expression in non-serotonergic/non-GABAergic neurons. Such changes in NMDA receptor expression in the PAG result in an alteration of the descending modulation of nociception, which might be an underlying mechanism for peripheral nerve injury-evoked persistent pain. Finally, the expression of NMDA receptors seems differentially regulated among neurons of different neurotransmitter phenotypes in the PAG.

Spinal Antiallodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice

Neuropathic pain is refractory against conventional analgesics, and thus novel medicaments are desired for the treatment. Glycinergic neurons are localized in specific brain regions, including the spinal cord, where they play an important role in the regulation of pain signal transduction. Glycine transporter (GlyT)1, present in glial cells, and GlyT2, located in neurons, play roles in modulating glycinergic neurotransmission by clearing synaptically released glycine or supplying glycine to the neurons and thus could modify pain signal transmission in the spinal cord. In this study, we demonstrated that i.v. or intrathecal administration of GlyT1 inhibitors, cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl methyl)amino methylcarboxylic acid (ORG25935) or sarcosine, and GlyT2 inhibitors, 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)-methyl]benzamide (ORG25543) and (O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-L-serine) (ALX1393), or knockdown of spinal GlyTs by small interfering RNA of GlyTs mRNA produced a profound antiallodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice. The antiallodynia effect is mediated through spinal glycine receptor alpha3. These results established GlyTs as the target molecules for the development of medicaments for neuropathic pain. However, these manipulations to stimulate glycinergic neuronal activity were without effect during the 4 days after nerve injury, whereas manipulations to inhibit glycinergic neuronal activity protected against the development of allodynia in this phase. The results implied that the timing of medication with their inhibitors should be considered, because glycinergic control of pain was reversed in the critical period of 3 to 4 days after surgery. This may also provide important information for understanding the underlying molecular mechanisms of the development of neuropathic pain.

Commonly Used Preclinical Models of Pain

Pain cuts across gender, age, and disease and is the most common reason people seek health-related treatment. Certain pain states do not respond to standard therapies, leaving nurses with few options to successfully care for patients in pain. Preclinical studies use many models to investigate the mechanisms and treatments for pain states similar to those encountered in humans. Within Cervero and Laird's conceptual framework of experimental pain, the authors present several commonly used preclinical models. Phase 1 pain models measure responses to a brief, controlled stimulus that minimizes tissue damage. Phase 2 pain models use topical or injected irritants, which cause inflammation and persistent pain that change peripheral and central neuronal responses. Phase 3 pain models produce neuropathic pain through partial or complete ligation of peripheral nerves. The use of preclinical models of pain can lead to the development of better pharmacological and nonpharmacological therapies that enhance nursing practice.

Enhancement of synaptic transmission and nociceptive behaviour in HPC‐1/syntaxin 1A knockout mice following peripheral nerve injury

Our previous analysis of HPC-1/syntaxin 1A knockout (KO) mice indicated that HPC-1/syntaxin 1A plays an important role in the synaptic plasticity of the hippocampus in vitro and learning behaviour in vivo. In order to gain further insights into the physiological functions of HPC-1/syntaxin 1A, we studied the changes in the plasticity of synaptic transmission in the superficial dorsal horn of the spinal cord following a peripheral nerve injury in HPC-1/syntaxin 1A KO and wild-type (WT) mice. The von Frey filament test revealed that partial ligation of the sciatic nerve caused neuropathic pain in both WT and KO mice. However, KO mice showed significant enhancement of mechanical allodynia as compared with WT mice. Tight-seal whole-cell recordings were obtained from neurons in the superficial dorsal horn of the spinal cord slices. Electrical stimulus-evoked excitatory postsynaptic currents (EPSCs), asynchronous EPSCs (aEPSCs) in the presence of strontium, and spontaneously occurring miniature EPSCs (mEPSCs) were analysed. Prior to peripheral nerve ligation, no significant differences were observed in the properties of evoked EPSCs, aEPSCs and mEPSCs in KO and WT mice. Seven-14 days after partial ligation, the amplitude of evoked EPSCs and the frequency of aEPSCs and mEPSCs in KO mice were significantly greater than those in WT mice; however, the amplitude of aEPSCs and mEPSCs remained unchanged in both groups. Enhanced allodynia behaviour and significant enhancement of excitatory synaptic transmission following peripheral nerve ligation in KO mice suggest that HPC-1/syntaxin 1A might play a role in synaptic plasticity in the nociceptive pathway.

Ciliary neurotrophic factor immunoreactivity is not associated with reactive astrocytes in the rat spinal cord following unilateral spinal nerve ligation

AbstractFollowing a direct insult to the central nervous system, ciliary neurotrophic factor (CNTF) is induced in reactive astrocytes adjacent to the injury, where it participates in functional recovery. On the other hand, the role of CNTF in spinal cord plasticity after peripheral nerve injury is unclear. Hence, CNTF immunoreactivity (IR) was evaluated in rat spinal cords following unilateral spinal nerve ligation. CNTF‐IR was found in GFAP‐negative white matter cells exhibiting oligodendrocyte‐like morphology. While GFAP labeling was increased in spinal cords of ligated animals, CNTF‐IR was unchanged, suggesting that CNTF does not regulate spinal astrocyte reactivity generated by peripheral nerve ligation.

Selective Reactions of Cutaneous and Muscle Afferent Neurons to Peripheral Nerve Transection in Rats

To determine whether peripheral nerve injury has similar effects on all functional types of afferent neuron, we retrogradely labeled populations of neurons projecting to skin and to muscle with FluoroGold and lesioned various peripheral nerves in the rat. Labeled neurons were counted after different periods and related to immunohistochemically identified ectopic terminals and satellite cells in lumbar dorsal root ganglia. After 10 weeks, 30% of cutaneous afferent somata labeled from transected sural nerves had disappeared but, if all other branches of the sciatic nerve had also been cut, 60% of cutaneous neurons were lost. Small-diameter sural neurons preferentially disappeared. In contrast, the number of muscle afferent somata was not affected by transection of various nerves. p75 was downregulated in axotomized cutaneous neurons but in not axotomized muscle neurons. Conversely, p75 was upregulated in satellite cells around cutaneous but not muscle neurons. Consistent with this, perineuronal rings containing tyrosine hydroxylase, calcitonin gene-related peptide, galanin, or synaptophysin were formed preferentially around cutaneous neurons. Selective lesions of predominantly cutaneous nerves triggered the formation of rings, but none were detected after selective lesions of muscle nerves. We conclude that cutaneous neurons are both more vulnerable and more associated with ectopic nerve terminals than muscle neurons in dorsal root ganglia after transection and ligation of peripheral nerves.

Contribution of the sensory and sympathetic nervous system to scalding-induced edema in the rat paw

It has recently been hypothesized that both the sensory and sympathetic nervous system contribute to the inflammatory reaction. A scalding model was developed in anaesthetized rats to investigate the contribution of neuropeptides in heat-induced edema localized to the hindpaw. After immersing the paw in water at 60°C for 10, 20, 30 and 60 s, edemic reactions were registered as change of paw volume in a plethysmograph and hindpaw perfusates collected to measure the content of neuropeptides by radioimmunoassay. A scalding period of 30 s induced the most prominent edemic reaction. There was a marked increase of the sensory neuropeptide neurokinin A and the sympathetic related transmitter neuropeptide Y in hindpaw perfusates after scalding. The effect of peripheral nerve ligation on edemic reaction and on the release of neuropeptides was investigated in rats scalded for 30 s at 60°C. There was a significant decrease of edema formation in the scalded nerve ligated paw as compared with the scalded paw on the non-ligated side. Neurokinin A was not detected in nerve ligated rats before or after scalding, whereas mononeuropathic rats showed increased concentrations of neuropeptide Y. The present results indicate that the sensory as well as the sympathetic nervous system, possibly through the release of neuropeptides, may contribute to scald-induced edema.

A simple systems approach to neural-immune communication.

The marine mollusc Aplysia californica is emerging as a useful model system to study neuralimmune communication at the mechanistic level because it has a well characterized nervous system that is easily accessible and it shares with mammals similar basic cellular defensive responses to wounded or non-self, i.e. the accumulation of defense cells (haemocytes) at the target site. Loose ligation of peripheral nerves in Aplysia induces a cellular defense response as evidenced by the accumulation of numerous haemocytes around the ligature. The excitability of nociceptive sensory neurons having axons close to the responding haemocytes is significantly increased. Haemocytes also accumulate at regions of axonal injury. The finding that human recombinant IL-1 beta can enhance the expression of injury-induced sensory hyperexcitability coupled with the detection of (ir)IL-1 in Aplysia haemolymph raises the interesting possibility that cytokines released from activated haemocytes attracted to an injured nerve or to a foreign body close to peripheral nerves may modulate nociceptive sensory function. The feasibility of using results from simple system such as Aplysia to formulate testable hypotheses in more complex systems is also discussed.

Effects of peripheral nerve ligation on expression of μ-opioid receptor in sensory ganglion neurons: an immunohistochemical study in dorsal root and nodose ganglion neurons of the rat

The present study was attempted to examine if μ-opioid receptor (MOR) might be transported by axonal flow peripherally through peripheral axons of somatic sensory ganglion neurons. After unilateral ligation of the sciatic nerve or the vagus nerve distal to the dorsal root ganglion (DRG) or nodose ganglion (NG), MOR-like immunoreactivity (MOR-LI) of neuronal cell bodies in the DRG of the fourth and fifth lumbar nerves, NG, ambiguus nucleus (Amb) and dorsal motor nucleus of the vagus nerve (DMV) on the side of the ligation was apparently reduced within 1 week after the nerve ligation. However, within 24 h after the nerve ligation, a transient enhancement of MOR-LI was observed in cell bodies of DRG neurons, sciatic nerve stump proximal to the ligature, and cell bodies of NG neurons on the side of the ligation; such a transient enhancement of MOR-LI was not detected in the Amb and DMV. The results suggest that MOR undergoes centrifugal axonal flow in peripheral axons of somatic and visceral sensory ganglion neurons, and that MOR synthesis in sensory ganglion neurons is vulnerable to damage of the peripheral axons.

KIF3A is a new microtubule-based anterograde motor in the nerve axon.

Neurons are highly polarized cells composed of dendrites, cell bodies, and long axons. Because of the lack of protein synthesis machinery in axons, materials required in axons and synapses have to be transported down the axons after synthesis in the cell body. Fast anterograde transport conveys different kinds of membranous organelles such as mitochondria and precursors of synaptic vesicles and axonal membranes, while organelles such as endosomes and autophagic prelysosomal organelles are conveyed retrogradely. Although kinesin and dynein have been identified as good candidates for microtubule-based anterograde and retrograde transporters, respectively, the existence of other motors for performing these complex axonal transports seems quite likely. Here we characterized a new member of the kinesin super-family, KIF3A (50-nm rod with globular head and tail), and found that it is localized in neurons, associated with membrane organelle fractions, and accumulates with anterogradely moving membrane organelles after ligation of peripheral nerves. Furthermore, native KIF3A (a complex of 80/85 KIF3A heavy chain and a 95-kD polypeptide) revealed microtubule gliding activity and baculovirus-expressed KIF3A heavy chain demonstrated microtubule plus end-directed (anterograde) motility in vitro. These findings strongly suggest that KIF3A is a new motor protein for the anterograde fast axonal transport.

Induction of a cellular defense reaction is accompanied by an increase in sensory neuron excitability in Aplysia

The complexity of vertebrate immune and nervous systems makes detailed cellular analysis of neuroimmune interactions a challenging prospect. The immune systems of invertebrates, although much less complex than their vertebrate counterparts, share basic cellular defense responses to wounded self or nonself. We have developed a simple model system to study neuroimmune interactions using an invertebrate preparation well suited to detailed cellular analysis. Loose ligation of peripheral nerves in Aplysia induced a cellular defense reaction evidenced by the accumulation of large numbers of amebocytes at the ligation site. From 5 to 30 d after ligation, the excitability of the soma of sensory neurons having axons in ligated nerves was increased compared to contralateral sensory neurons with axons in nonligated nerves. Spike threshold and afterhyperpolarization were reduced, and spike amplitude and duration were increased. Spike accommodation was also decreased such that sensory neurons on the ligated side fired more spikes to a 1 sec intracellular depolarizing pulse than control sensory neurons. These effects are unlikely to be accounted for by ligation-induced injury of sensory axons since both morphological and electrophysiological evidence indicated that the axons in ligated nerves were healthy and able to conduct action potentials. Amebocytes activated by the presence of nonself may release factors that lead to a central sensitization of sensory neurons with axons in close proximity to the amebocytes.

Induction of a cellular defense reaction is accompanied by an increase in sensory neuron excitability in Aplysia.

The complexity of vertebrate immune and nervous systems makes detailed cellular analysis of neuroimmune interactions a challenging prospect. The immune systems of invertebrates, although much less complex than their vertebrate counterparts, share basic cellular defense responses to wounded self or nonself. We have developed a simple model system to study neuroimmune interactions using an invertebrate preparation well suited to detailed cellular analysis. Loose ligation of peripheral nerves in Ap/ysia induced a cellular defense reaction evidenced by the accumulation of large numbers of amebocytes at the ligation site. From 5 to 30 d after ligation, the excitability of the soma of sensory neurons having axons in ligated nerves was increased compared to contralateral sensory neurons with axons in nonligated nerves. Spike threshold and afterhyperpolarization were reduced, and spike amplitude and duration were increased. Spike accommodation was also decreased such that sensory neurons on the ligated side fired more spikes to a 1 set intracellular depolarizing pulse than control sensory neurons. These effects are unlikely to be accounted for by ligation-induced injury of sensory axons since both morphological and electrophysiological evidence indicated that the axons in ligated nerves were healthy and able to conduct action potentials. Amebocytes activated by the presence of nonself may release factors that lead to