Specificity of Androgenic Regulation of Input to Sexually Dimorphic Motoneurons in Male Rat Lumbar Spinal Cord

Averaged antidromic field potentials were recorded near motoneuronal somata of the spinal nucleus of the bulbocavernosus (SNB) after stimulation of their axons, in intact, castrated, and testosterone-treated castrated male rats under urethane anesthesia. When the SNB motoneurons were antidromically activated with double pulses to the bulbocavernosus (BC) nerve, the first pulse inhibited the antidromic response caused by the second pulse at interstimulus intervals of 5-40 ms, implying the presence of recurrent or other inhibition activated by BC nerve stimulation. Castration and androgen treatment had no significant effect on the amplitude of the suppression. In this regard this pathway differs from other pathways that synapse onto SNB motoneurons, in which activity is highly sensitive to androgen, indicating that androgen exerts its effects only on specific neural circuits that influence SNB motoneurons.     

Spinal Cord Stimulation for the Treatment of Chronic Pain in Patients with Lumbar Spinal Stenosis

Objective: Chronic back and leg pain associated with lumbar spinal stenosis (LSS) is common in the elderly. Surgical decompression is usually performed when conservative treatments fail. We present an evaluation of the long‐term outcome of patients suffering from symptomatic LSS treated with spinal cord stimulation (SCS). Materials and Methods: Data were collected prospectively in three independent registries in three European centers. Pooled data were analyzed retrospectively. Changes in pain intensity, functional status, and analgesic medication were compared at baseline and at the last available follow‐up. Demographic data as well as details regarding the implantation procedure and any adverse events were systematically recorded. Results: Data were recorded in 69 patients with a mean follow‐up period of 27 months. All patients showed clinically and statistically significant improvement in pain relief, the visual analog scale decreasing from 7.4 ± 2.3 to 2.8 ± 2.4 (p < 0.05). The use of analgesic medication decreased and the functional status improved. Conclusion: Spinal cord stimulation seems to be effective in the treatment of patients suffering from chronic pain associated with LSS. Being less invasive and reversible, SCS should be considered before surgical decompression, particularly in patients with increased risks associated with back surgery.     

Galanin Receptor Binding Sites in Adult Rat Spinal Cord Respond Differentially to Neonatal Capsaicin, Dorsal Rhizotomy and Peripheral Axotomy

The discrete distribution and possible changes in specific [¹²⁵I]galanin binding sites were evaluated in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. The highest density of [¹²⁵I]galanin binding sites in the normal rat spinal cord was particularly evident in the superficial layers of the dorsal horn whereas moderate to low amounts of labelling were associated with the deeper dorsal horn, areas around the central canal and the ventral horn. Capsaicin-treated rats, compared to littermate controls, showed a significant bilateral increase in [¹²⁵I]galanin binding in the superficial laminae of the dorsal horn. Similarly, unilateral dorsal rhizotomy evoked a significant increase in the density of [¹²⁵I]galanin binding sites in the superficial dorsal horn ipsilateral to surgery. Section of the sciatic nerve, on the other hand, induced a significant depletion in [¹²⁵I]galanin binding in laminae I and II of the ipsilateral dorsal horn. These results, in parallel to those reported for galanin immunoreactivity under similar conditions, suggest that [¹²⁵I]galanin binding sites are preferentially located postsynaptically to the primary afferent fibre terminals in the dorsal horn of the spinal cord. Thus it seems that galanin, at the level of the dorsal spinal cord, regulates the processing of nociceptive information by acting on its own class of specific receptors located postsynaptically to primary sensory terminals.     

Autoradiographic Study of Binding and Internalization of a Luteinizing Hormone-Releasing Hormone Antagonist [D-Nal1, D-Cpa2, A-D-Trp3, D-Arg6, D-Ala10]LHRH by Rat Pituitary Gonadotrophs

The binding and intracellular pathway of the radioiodinated luteinizing hormone-releasing hormone (LHRH) antagonist [D-Nal¹, D-Cpa², D-Trp³, D-Arg⁶, D-Ala¹⁰]LHRH in pituitary gonadotrophs was studied as a function of time after iv injection of label into intact and castrated female rats. In semithin (1 |im) sections, silver grains were exclusively localized over about 10% of anterior pituitary cells in intact animals. In castrated animals, only the large castration cells were labeled. In control rats injected with both iodinated antagonist and an excess of unlabeled peptide, no significant labeling could be detected. At the ultrastructural level, the silver grains were exclusively localized in gonadotrophic cells. The time-course study showed that 30 min and 60 min after injection a high proportion of the silver grains was associated with the plasma membrane. Six h after injection, an appreciable proportion of the label was found over intracellular organelles, especially lysosomes and secretory granules. These results indicate that the potent LHRH antagonist used in the present experiments binds selectively to gonadotrophs and is subsequently internalized but at a much lower rate than that observed with LHRH agonists. This slow internalization of the LHRH antagonist might be related to the normal endocytic processes which occur independently of receptor activation.     

Postischemic changes of [3H]nimodipine and [3H]ryanodine binding in the gerbil striatum and hippocampus

Sequential alterations of [³H]nimodipine and [³H]ryanodine binding in gerbils were investigated in selectively vulnerable regions, such as the striatum and hippocampus, 1 h to 7 days after 10 min of transient cerebral ischemia. [³H]Nimodipine binding showed no significant changes in the striatum and hippocampus up to 48 h after ischemia. Seven days after ischemia, however, a severe reduction in [³H]nimodipine binding was observed in the dorsolateral striatum, hippocampal CA1 (stratum oriens, stratum pyramidale and stratum radiatum) and hippocampal CA3 sector. On the other hand, [³H]ryanodine binding showed a significant increase in the hippocampus 1 h after ischemia. Five hours after ischemia, a significant reduction in [³H]ryanodine binding was observed only in the hippocampal CA1 sector. Thereafter, the striatum and hippocampus showed no significant alterations in [³H]ryanodine binding up to 48 h after ischemia. After 7 days, a marked reduction in [³H]ryanodine binding was observed in the striatum and hippocampus which were particularly vulnerable to ischemia. These results demonstrate that postischemic alteration in [³H]nimodipine and [³H]ryanodine binding is produced with different processes in the hippocampus. They also suggest that the mechanism for striatal cell damage caused by transient cerebral ischemia may, at least in part, differ from that for hippocampal neuronal damage. Furthermore, our findings suggest that abnormal calcium release from intracellular stores may play a pivotal role in the development of hippocampal neuronal damage.     

Local Transcutaneous Cooling of the Spinal Cord in the Rat: Effects on Long-Term Outcomes After Compression Spinal Cord Injury

This study tested efficiency of a novel thermoelectric cooler for local transcutaneous spinal cord cooling. Spinal cord compression was made by epidural balloon inflation at T8-T9 level of the spinal cord. Experimental animals (n = 20) were divided into two groups. In the hypothermic group, local cooling started 25 min after spinal cord injury and lasted for 1 h with paravertebral temperature maintained at 28.5°C (±0.3). Normothermic group underwent identical procedures, but their temperature was maintained normothermic. The assessment of neurologic recovery was performed once a week during a 4 weeks survival period. After 4 weeks animals were sacrificed and the extent of the spinal cord lesion morphometrically evaluated. There were no statistically significant intergroup differences in BBB scores and preserved volumes of the spinal cord tissue. In consecutive cross-sectional areas, hypothermic animals had significantly more preserved white matter at the cranial periphery of the lesion. It was concluded that mild posttraumatic hypothermia (31.8°C) had some protective effect on tissue loss after spinal cord injury but this effect was not associated with functional improvement.     

Activation by Cutaneous or Visceral Noxious Stimulation of Spinal Neurons Projecting to the Medullary Dorsal Reticular Nucleus in the Rat: A c-fos Study

The involvement of spinal neurons in the transmission of cutaneous and visceral nociceptive input to the medullary dorsal reticular nucleus was studied. Rats were injected with cholera toxin subunit B in the left dorsal reticular nucleus and subjected 4 days later to noxious mechanical, thermal or chemical stimulation of the proximal internal aspect of the left thigh, or to chemical stimulation of the urinary bladder. Sections of spinal segments T₁₃-L₃ were processed immunocytochemically for cholera toxin subunit B and Fos protein. The percentage of double-labelled cells in the population of Fos-positive cells was higher in lamina I (1–4%) than in deeper laminae (0–0.7%) following all stimuli. The percentage of double-labelled cells in the population of retrogradely labelled cells was 30–53% in lamina I and 0–5% in laminae III—X. Visceral stimulation activated more retrogradely labelled lamina I cells than any kind of cutaneous stimulation. Pyramidal cells were activated in higher numbers than multipolar and flattened cells after thermal cutaneous or visceral stimulation, and in lower numbers than multipolar cells after mechanical stimulation. These results suggest that, in the experimental conditions used, spinal cord cells conveying noxious input to the dorsal reticular nucleus are concentrated in lamina I. They further indicate that the spinaldorsal reticular nucleus pathway plays a major role in the transmission of nociceptive visceral input, and point to the preferential involvement of pyramidal cells in cutaneous thermal and visceral processing.     

Membrane Characteristics and Synaptic Responsiveness of Superficial Dorsal Horn Neurons in a Slice Preparation of Adult Rat Spinal Cord

Intracellular recordings have been made from neurons of the superficial dorsal horn in slices of the lumbar and thoracic spinal cord of young adult rats. Three broad categories of neurons could be distinguished on the basis of their firing patterns to intracellular current pulses and their afterhyperpolarizations (AHP); there was no detectable difference in the regional distribution of the three types. Category 1 cells were characterized by maintained firing to intracellular depolarizing current pulses, brief action potential durations and polyphasic AHPs. Category 2 cells showed spike adaptation, without spike attenuation, during intracellular current pulses, and had monophasic AHPs. Category 3 cells fired only 1 or 2 spikes to maintained depolarizing pulses and had smaller monophasic AHPs than category 2 neurons. Spontaneous excitatory and inhibitory postsynaptic potential (epsp and ipsp) activity was seen with psp durations varying widely. Low intensity electrical stimulation of afferent fibres, or of superficial white matter, resulted in polyphasic epsps and/or ipsps. The spike discharge in response to such afferent inputs correlated with the membrane properties of the cells, such that the synaptic responses of category 1 neurons were usually bursts of spikes, whereas category 2 and 3 neurons either failed to fire or fired only a single spike. These results in adult rat spinal cord suggest that the discharge pattern within synaptic sensory responses of superficial dorsal horn neurons is determined by postsynaptic membrane properties as well as by the pattern of the afferent input.     

Cellular and Subcellular Distribution of NMDAR1 Splice Variant mRNA in the Rat Lumbar Spinal Cord

The regional distribution of alternatively spliced messenger RNA encoding the N -methyl-D-aspartate (NMDA) receptor R1 subunit (NMDAR1) variants was examined by in situ hybridization in the rat lumbar spinal cord. Splice-specific oligonucleotide probes [recognizing full-length mRNA (NMDAR1-1), deletion exon 21 (NMDAR1-2), deletion exon 22 (NMDAR1-3), combined deletion exons 21 and 22 (NMDAR1-4) and mRNA which lacks (NMDAR1-a) or contains exon 5 (NMDAR1-b)] detected marked differences in abundance and distribution of N- and C-terminal spliced variants. The NMDAR1-a, NMDAR1-2 and NMDAR1-4 mRNAs were evenly distributed throughout all laminae of the dorsal and ventral horns. In the superficial dorsal horn NMDAR1-b mRNA was preferentially detected in laminae II inner and III, while NMDAR1-1 mRNA was restricted to laminae I to III. Large neurons in laminae IV and V contained mainly NMDAR1-a, NMDAR1-2 and NMDAR1-4 mRNAs and occasionally NMDAR1-b. The NMDAR1-3 variant was only detected in very low abundance, being restricted to occasional cells in lamina I and II. In the ventral horn, motor neurons showed strong signals for NMDAR1-a, NMDAR1-b, NMDAR1-2 and NMDAR1-4 mRNAs. Serial sectioning through large motor neurons permitted the detection of multiple splice variants in single neurons. Analysis of the subcellular distribution of the mRNAs revealed that the NMDAR1-1 mRNA was almost exclusively found in the cell nucleus, NMDAR1-a mRNA was largely in the cytoplasm, while all other splice variants showed a homogeneous distribution between nucleus and cytoplasm. Comparison of the in situ hybridization images with functional analyses of heteromeric recombinant receptors will be necessary to ascertain whether splice variants of the NMDAR1 receptor subunit can account for some of the known electrophysiological properties of spinal cord neurons under physiological and pathophysiological conditions.     

Spinal Cord Stimulation for the Treatment of Upper and Lower Extremity Neuropathic Pain due to Lyme Disease

Background. Lyme disease is caused by Borrelia, a bacterium transmitted by the bite of a deer tick. A slow developing encephalopathy or an axonal polyneuropathy with distal paresthesia and spinal or radicular pain rarely occurs and can be hard to treat. Materials and Methods. We report here the case of a 44‐year‐old woman with four‐limb, intolerable, neuropathic pain as sequelae to Lyme disease, which was resistant to conservative measures and was treated successfully with concurrent, thoracic, and cervical percutaneous spinal cord stimulation (SCS). Results. After 18 months of therapy and follow‐up, this patient's analgesia, as a result of SCS, continues to be excellent, with almost complete subjective pain relief and cessation of adjuvant analgesic medication. Conclusions. SCS may be efficacious for the treatment of neuropathic pain due to Lyme disease.     

Developmental Loss of GABA- and Glycine-induced Depolarization and Ca2+ Transients in Embryonic Rat Dorsal Horn Neurons in Culture

More than 90% of dorsal horn neurons from embryonic day 15–16 rats responded to the inhibitory amino acids GABA and glycine by a transient elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) when maintained in culture for <1 week. This [Ca²⁺]_i response has previously been shown to be due to depolarization and subsequent Ca²⁺ entry through voltage-gated Ca²⁺ channels following activation of bicuculline-sensitive GABA_A receptors and strychnine-sensitive glycine receptors. Both the number of cells responding to GABA and glycine and the amplitude of the [Ca²⁺]_i response diminished over time in culture. By 30 days in culture, none of the cells responded to GABA, muscimol or glycine by elevation of [Ca²⁺]_i. The loss of the [Ca²⁺]_i response was not due to a change in the abundance or the properties of voltage-gated Ca²⁺ channels, since over the same period of time dorsal horn neurons showed a large increase in the amplitude of the [Ca²⁺]_i transient in response to 30 mM K⁺. Nor was the loss of the [Ca²⁺]_i response due to a loss of GABA and glycine receptors. Instead, the decrease in the [Ca²⁺]_i response over time paralleled a similar change in the electrophysiological responses. More than 90% of the neurons tested were depolarized in response to inhibitory amino acids during the first week in culture. After 30 days, all neurons tested responded to GABA and glycine with a hyperpolarization. These observations add support to the suggestion that GABA and glycine may excite dorsal horn neurons earlyin development and play a role in postmitotic differentiation.     

Active and Passive Membrane Properties of Spinal Cord Neurons that Are Rhythmically Active during Swimming in Xenopus Embryos

Cellular properties have been examined in ventrally located Xenopus spinal cord neurons that are rhythmically active during fictive swimming and presumed to be motoneurons. Resting potentials and input resistances of such neurons are - 75 +/- 2 mV (mean +/- standard error) and 118 +/- 17 M ohm respectively. Most cells fire a single impulse, 0.5 to 2.0 ms in duration and 48.5 +/- 1.8 mV in amplitude, in response to a depolarizing current step. A minority fire several spikes of diminishing amplitude to more strongly depolarizing current. Cells held above spike, threshold fire on rebound from brief hyperpolarizing pulses. Spikes are blocked by 0.1 to 1.0 microM tetrodotoxin (TTX) and are therefore Na+-dependent. Current/voltage (I/V) plots to injected current are approximately linear near the resting potential but become non-linear at more depolarized levels. Cells recorded in TTX with CsCI-filled microelectrodes show a linearized I/V plot at depolarized membrane potentials suggesting the normal presence of a voltage-dependent K+ conductance activated at relatively depolarized levels. Most cells recorded in this way but without TTX fire long trains of spikes of near constant amplitude, pointing to a role of the K+ conductance in limiting firing in normal cells. Spike blockage with TTX reveals, in some cells, a transient depolarizing Cd2+-sensitive and therefore presumably Ca2+-dependent potential that increases in amplitude with depolarization. Cells in TTX, Cd2+, and strychnine, and recorded with CsCI-filled microelectrodes to block active conductances respond to hyperpolarizing current steps with a two component exponential response. The cell time constant (tau0) obtained from the longer of these by exponential peeling is relatively long (mean 15.7 ms). These findings contribute to an increased understanding of the cellular properties involved in spinal rhythm generation in this simple vertebrate.     

Corticotropin-Releasing Hormone Neurons in the Paraventricular Nucleus Project to the External Zone of the Median Eminence: A Study Combining Retrograde Labeling with Immunocytochemistry

Corticotropin-releasing hormone (CRH) is the major regulator of the pituitary-adrenal axis. CRH-immunoreactive perikarya are widely distributed in the central nervous system; however, only those which participate directly in the regulation of adrenocorticotropin are connected to the portal circulation in the external zone of the median eminence. The present study describes the identification of these hypophysiotropic neurons using retrograde labeling and CRH immunocytochemistry. Fuoro-Gold was injected peripherally then, 5 days later, the animals were treated with colchicine. Twenty-four hours later the animals were sacrificed, and their brains were immunostained for CRH with the indirect immunofluorescence technique. The results indicate that the vast majority of the Fluoro-Gold-accumulating and CRH-immunopositive perikarya (hypophysiotropic neurons) are located in the medial parvicellular subdivision of the paraventricular nucleus (PVN). However, not each CRH-immunoreactive neuron contains Fluoro-Gold, i.e. a small portion of these neurons project to areas of the brain other than the median eminence. The anterior, lateral and periventricular subdivisions of the PVN also contain hypophysiotropic CRH-immunoreactive perikarya, however, their number is much less than in the medial parvicellular subdivision. Scattered double-labeled cells are also present in the medial preoptic area and the dorsal hypothalamus, just behind the PVN. These results support previous observations that the PVN, particularly the medial parvicellular subdivision, is the predominant source of the hypophysiotropic CRH neurons.     

Colocalization of Oestrogen Receptor lmmunoreactivity and Preproenkephalin mRNA Expression to Neurons in the Superficial Laminae of the Spinal and Medullary Dorsal Horn of Rats

A double-labelling procedure combining immunohistochemical staining with in situ hybridization using a radiolabelled cRNA probe was employed to demonstrate oestrogen receptor-like immunoreactivity and preproenkephalin-A mRNA in the medullary and spinal dorsal horn of female rats. Both markers labelled large numbers of neurons in the substantia gelatinosa and its trigeminal homologue. Many of these neurons were double-labelled, displaying both oestrogen receptor-like- immunoreactivity and preproenkephalin-A mRNA; cell counts showed that 40–60% of the of the oestrogen receptor-like-immunoreactive cells in the superficial laminae also were labelled for preproenkephalin-A mRNA, and that 60–70% of the preproenkephalin-A mRNA-labelled neurons in the same laminae displayed oestrogen receptor-like immunoreactivity. Previous studies have shown that oestrogen receptors can bind to the promoter region of the preproenkephalin-A gene, and studies on the hypothalamus have demonstrated that oestrogen regulates enkephalin expression in select neuronal populations. The present results demonstrate that enkephalinergic neurons in the superficial dorsal horn contain oestrogen receptors and suggest that oestrogen may play an important role in the modulation of sensory and nociceptive processing in the lower medulla and spinal cord.     

Formation of Neurons in the Sexually Dimorphic Anteroventral Periventricular Nucleus of the Preoptic Area of the Rat: Effects of Prenatal Treatment with Testosterone Propionate

An examination was made of neurogenesis in the anteroventral periventricular nucleus (AVPv) of the preoptic area of the rat using bromodeoxyuridine (BrdU), a thymidine analog, and a BrdU-specific antibody. Cells in the AVPv of adult rats were labeled with the antibody when BrdU was injected into pregnant rats once during day 13 to 18 of gestation, but not during day 10 to 12 nor 19 to 20 of gestation nor on postnatal day 1, indicating that neurogenesis of the AVPv occurs during a limited period from day 13 to 18 of gestation. Next, to examine the effects of androgen on neurogenesis, BrdU was injected once on day 15 into pregnant rats that also received injections of testosterone propionate (TP). The number of BrdU-labeled cells in the AVPv was similar in control female and male fetuses and female fetuses from pregnant rats that received daily injections of TP during days 14 to 16, when fetuses were examined on day 17 of gestation. These results suggest that the neurogenesis that was recognized by labeling with BrdU was not affected by the treatment with TP. On day 21 of gestation, BrdU-labeled cells in the AVPv of control male fetuses and female fetuses that received TP during days 14 to 18 were fewer in number than those in female fetuses of the control group, whereas treatments with TP during days 14 to 16 and during days 17 to 18 did not cause any significant decrease in number of BrdU-labeled cells. These findings support the hypothesis that elimination of a population of cells, for example, by cell death as described previously, is enhanced in male fetuses and in female fetuses treated with TP repetitively.     

Vasopressin Anti-Idiotypic Antibody Staining in the Rat Brain: Colocalization with [35S][pGlu4, Cyt6]AVP(4–9) Binding Sites

Vasopressin and its fragment peptides such as [pGlu⁴, Cyt⁶]AVP_(4–9) (AVP_(4–9)) represent putative neuromodulators within central nervous homeostatic, memory and behavioural circuits. To localize their central receptor systems, the previously characterized monoclonal anti-idiotypic antibody mAb 237 was employed in immunocytological investigations of rat brain tissue sections. This antibody was raised to the monoclonal idiotypic anti-AVP antibody mAb 113 which preferentially binds to the acyclic C-terminal portion of the AVP molecule and is therefore also capable of binding the naturally occurring AVP_(4–9) fragment. Immunoreactive magnocellular neurones were detected in the AVP-synthesizing supraoptic but not paraventricular nuclei. Dense staining was observed within circumventricular organs lacking a blood-brain barrier (BBB). These structures include the subfornical organ, the organum vasculosum laminae terminalis, the internal layer of the median eminence, the body of the pineal gland, the choroid plexus and the area postrema, where immunoreactivity was found on capillaries, neurones and fibres. Further staining was found in the nucleus of the solitari tract and the arcuate nucleus, endowed with a leaky BBB. Distinct cell patches in the ependymal lining of the third ventricle as well as dendritic processes of juxtaependymal neurones were labelled by the anti-idiotypic antibody mAb 237. The observed staining pattern did not parallel that obtained in autoradiographic studies performed using either radiolabelled AVP or a V₁-receptor antagonist, but that found with the [³⁵S]-labelled AVP_(4–9) fragment. Using [³⁵S]-labelled AVP_(4–9) fragment, specific high density binding sites could be localized autoradiographically in structures within and outside the BBB, in complete agreement with the anti-idiotypic immunoreactivity. Since the anti-idiotypic methodology is based on transfer of complementary structures, and the epitope recognized by the corresponding idiotypic antibody resembles the sequence of AVP_(4–9), the anti-idiotypic antibodies might recognize the AVP_(4–9) receptor with high affinity.