Ultrastructural localization of beta-arrestin-1 and -2 in rat lumbar spinal cord.

beta-arrestins play significant roles in agonist-mediated desensitization of G protein-coupled receptors. Although the presence of beta-arrestin subtypes, beta-arrestin-1 and(- 2) in rat brain has been studied extensively, their existence in the spinal cord has not been described. In the current study, we performed immunohistochemical analyses of beta-arrestins at both light and electron microscopic levels using rat lumbar 1-2 spinal cord segments. Intense immunoreactivity for beta-arrestin-1 was found in the motoneurons in lamina IX of the ventral horn and elongated cells in the dorsal nucleus of Clarke. Modest immunoreactivity was detected among the neurons of laminae V and VII/VIII, and weaker immunoreactivity in laminae III, IV, and X. beta-arrestin-2 immunoreactivity was also distributed through laminae III-X in the order of IX > dorsal nucleus of Clarke > V > VII/VIII > IV > III > X. Laminae I and II did not show immunoreactivity. At the electron microscopic level, both beta-arrestin-immunoreactive and nonimmunoreactive dendrites were observed, whereas axons and terminal boutons were devoid of immunoreactivity. In immunoreactive dendrites most beta-arrestin immunoreactivity was distributed throughout the cytoplasm, demonstrating their association with microtubules. In addition, strong immunoreactivity was often found at postsynaptic densities. Our results thus suggest beta-arrestins' possible involvement in both motor and sensory mechanisms at the postsynaptic level in rat lumbar spinal cord.     

Quantitative autoradiographic distribution of calcitonin gene-related peptide (hCGRP alpha) binding sites in the rat and monkey spinal cord.

Calcitonin gene-related peptide (CGRP) has been implicated in various spinal functions on the basis of its presence in the substantia gelatinosa and motoneurons and the biological effects induced by intrathecal CGRP injections. We investigated here the comparative distribution of [125I]hCGRP alpha binding sites in various segments of the rat and monkey spinal cord. The immunocytochemical localization of CGRP-like material in rat spinal cord was also evaluated for comparison. In the rat spinal cord, high densities of [125I]hCGRP alpha binding sites were observed in lamina I, in a U-shaped band that included lamina X and the medial parts of laminae III-IV and in the intermediolateral and intermediomedial nuclei. The substantia gelatinosa (lamina II) contained relatively lower, but still significant, densities of [125I]hCGRP alpha binding sites, while the ventral horn showed low amounts of specific labeling. CGRP-like immunoreactive fibers, on the other hand, were heavily concentrated in laminae I-II and in the reticulated portion of lamina V of the dorsal horn. Immunoreactivity to CGRP antiserum was also noted in fibers around the central canal and in a number of motoneurons of the ventral horn. In the monkey spinal cord, [125I]hCGRP alpha binding sites were present in lamina I in a U-shaped band that included lamina X and the medial portions of laminae V-VI. Relatively low levels of [125I]hCGRP alpha binding were detected in laminae II to IV of the dorsal horn, while the ventral horn was more enriched with specific [125I]hCGRP alpha binding sites. Thus, it appears that the autoradiographic distribution of [125I]hCGRP alpha sites is species dependent in the spinal cord. Additionally, some differences are observed between the localization of [125I]hCGRP alpha binding sites and immunoreactive material in the rat spinal cord. These differences may be relevant to the purported roles of CGRP-like peptides in spinal functions such as nociception, control of sympathetic output, and motor control.     

The autoradiographic localization of substance P receptors in the rat and bovine spinal cord and the rat and cat spinal trigeminal nucleus pars caudalis and the effects of neonatal capsaicin

Substance P (SP) is a putative neurotransmitter in the central nervous system. In the present report we have used autoradiographic receptor binding techniques to investigate the distribution of SP receptor binding sites in the rat and bovine spinal cord and in the rat and cat spinal trigeminal nucleus pars caudalis. Although some quantitative differences were evident, all species appeared to have a similar distribution of SP receptor binding sites in both the spinal cord and in the spinal trigeminal nucleus pars caudalis. In the spinal cord the heaviest concentration of SP receptors is located in lamina X, while moderate to heavy concentrations were found in laminae I, II and V-IX. Very low concentrations of SP receptors were present in laminae III and IV. Examination of the cat and rat spinal trigeminal nucleus pars caudalis revealed a moderate density of SP receptor binding sites in laminae I and II, very low concentrations in laminae III and IV, and low to moderate concentrations in lamina V. Rats treated neonatally with capsaicin showed a small (11%) but significant (P < 0.02) increase in the levels of SP receptor binding sites in laminae I and II of the cervical and lumbar spinal cord while in all other laminae the levels remained unchanged.     

Alterations in receptors for thyrotropin-releasing hormone, serotonin, and acetylcholine in amyotrophic lateral sclerosis

We utilized quantitative autoradiography to examine thyrotropin-releasing hormone (TRH) receptors, serotonin type 1A (5-HT1A) receptors, muscarinic cholinergic receptors, choline uptake sites, beta-adrenergic receptors, and norepinephrine uptake sites in discrete laminae of spinal cord from patients with amyotrophic lateral sclerosis (ALS) and non-neurologic controls. We found decreases of over 50% in the concentration of TRH receptors in lamina IX of cervical, thoracic, and lumbar spinal cord from ALS patients. Similar reductions were noted in concentrations of muscarinic cholinergic receptors in lamina IX of spinal cords from ALS patients. Significant increases of up to 140% in 5-HT1A receptor densities were noted in lamina IX of spinal cords from ALS patients. No differences were noted between the concentrations of beta-adrenergic receptors or norepinephrine uptake sites in patients with ALS and controls. These findings suggest that TRH and 5-HT may be involved in the pathophysiology of ALS, and act in a comodulatory role in the normal spinal cord.     

Impact of food deprivation on alpha 1- and alpha 2-noradrenergic receptors in the paraventricular nucleus and other hypothalamic areas

Hypothalamic norepinephrine may modulate normal eating behavior through activation of alpha 2-noradrenergic receptors, localized in the paraventricular nucleus (PVN). We investigated whether these receptors, which stimulate food ingestion, may in turn be altered by the nutritional state of the organism. Thus the impact of food deprivation, on the specific binding of [3H]-p-aminoclonidine ([3H]PAC) to alpha 2-noradrenergic receptors in discrete hypothalamic areas, was examined in rats. The results of our first experiment revealed that 48 hr food deprivation reduced (by 50%) the maximum number of binding sites (Bmax) of the high affinity component of [3H]PAC binding to alpha 2 receptors. This effect occurred exclusively in the medial hypothalamus (which includes the PVN), without any change in the affinity (Kd) of these receptors. A smaller decline was seen in the low affinity binding sites of the medial hypothalamus, whereas no changes were observed in the density or affinity of the high and low affinity alpha 2 receptor sites in the lateral hypothalamus or frontal cortex. The alpha 1-noradrenergic receptor sites, as defined by [3H]prazosin and [3H]WB-4101 binding, were also unaffected in the different brain areas by 48 hr food deprivation. An additional analysis of alpha 2 receptors in discrete hypothalamic nuclei demonstrated that the deprivation-induced decline in alpha 2-receptor binding: occurred specifically in the PVN; was apparent after as little as 3 hr food deprivation; and occurred only when this brief deprivation fell at the onset of the dark cycle, as opposed to at the end of the dark cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

Autoradiographic analysis of 125I-substance P binding in rat spinal cord following chronic constriction injury of the sciatic nerve.

Using receptor binding and autoradiographic techniques, changes in Bolton-Hunter labeled 125I-substance P (125I-BH-SP) binding were determined in laminae I/II, V and X of rat lumbar spinal cord after chronic constriction injury (CCI) of the sciatic nerve. When compared to the sham-operated side of the control group, SP binding significantly increased ipsilateral to the CCI in laminae I/II at 5, 10 and 20 days after injury and in lamina V at 5 days after injury. Scatchard analysis was performed on the 125I-BH-SP binding to the NK1 receptor in laminae I/II of rats 5 days after generation of the CCI. A significant decrease in the Kd of 125I-BH-SP binding was observed in laminae I/II ipsilateral to CCI when compared with the control side (ipsilateral to sham surgery). There was no significant change in the Bmax in laminae I/II ipsilateral to CCI. The changes in 125I-BH-SP binding in the rat spinal cord that occurred after CCI were found in areas of the spinal cord that receive terminations of nociceptive primary afferent fibers. The increased affinity of the NK1 binding site that we report could result in an increase in SP receptor activation in laminae I/II. Such central changes in SP binding may contribute to the neuropathic pain syndrome observed in rats with the CCI.     

The 5-HT2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons

Serotonin (5-HT) plays a major role at the spinal level by modulating most spinal functions through several receptor subtypes including the 5-HT2A receptor. To gain further insight into the cellular role of this receptor, we performed an immunocytochemical study of 5-HT2A receptors in the rat spinal cord, at light and electron microscope levels. The results showed that 5-HT2A receptors were widely distributed in the spinal cord at all segmental levels. Immunolabeling was particularly dense in lamina IX and in the dorsal horn lamina IIi. Immunoreactive cell bodies were numerous in lamina IX, where many but not all motoneurons were labeled, as shown by double labeling with choline acetyltransferase antibodies. Stained cell bodies were also observed in the gray matter. The study at the ultrastructural level focused on the lumbar dorsal horn (laminae I-II) and ventral horn (lamina IX). At both levels, 5-HT2A immunoreactivity was mainly postsynaptic on dendrites and cell bodies. However, a little presynaptic labeling was also observed in axon and axon terminals, some of them containing large granular vesicles attesting to their peptidergic nature. The main result of our study was the "nonsynaptic" plasma membrane localization of 5-HT2A receptors covering a large surface of cell bodies and dendrites, suggesting a paracrine form of action of serotonin. These observations are consistent with a double role (pre- and postsynaptic) for serotonin on these receptors on various cellular targets.     

Quantitative autoradiographic mapping of a novel serotonin receptor-5-HT1S.

The spinal cord distribution of a recently identified, high affinity 3H-5-HT binding site, the 5-HT1S receptor was studied employing quantitative autoradiography with microdensitometry. The laminar distribution and density of other high affinity 3H-5-HT binding sites present in rat spinal cord, 5-HT1A, 5-HT1B and 5-HT1C, was also determined. The greatest density of 5-HT1S receptors was found in the superficial dorsal horn laminae, the autonomic nuclei around the central canal and the ventral motor nuclei. Smaller, variable densities of 5-HT1S receptors were observed in other spinal cord regions. A similar spatial pattern of receptor density was observed for 5-HT1A, 5-HT1B and 5-HT1C receptors. The distribution of 5-HT1S receptors suggests functional modulation of somatosensory input to spinal cord as well as control of visceral and somatomotor activity.     

Diffuse distribution of sulforhodamine-labeled neurons during serotonin-evoked locomotion in the neonatal rat thoracolumbar spinal cord

The fluorescent dye sulforhodamine-101 undergoes synaptic activity-dependent endocytotic uptake and consequent retrograde transport in presynaptic neurons. We used sulforhodamine to identify thoracolumbar spinal premotor neurons (T11-L6) activated during serotonin (5-HT) -induced hindlimb locomotor-like activity in the in vitro neonatal rat spinal cord preparation. Sulforhodamine labeling required locomotor-like activity because few neurons were labeled unless bath applied 5-HT recruited the locomotor rhythm. In contrast, N-methyl-D-aspartate (NMDA; 5 microM) profoundly increased spinal neuronal labeling irrespective of locomotor activity. The contribution of false-positive activity labeling during locomotion induced by application of NMDA with 5-HT (Kjaerulff et al. [1994] J Physiol (Lond). 478:265-273) necessitated the present re-mapping of sulforhodamine-labeled neurons. During 5-HT-evoked locomotion, the sulforhodamine-labeled neurons were diffusely scattered within the spinal cord with predominant labeling in lamina VII. Motor nuclei (lamina IX) and superficial laminae (I-II) were typically devoid of labeled cells in the isolated spinal cord. However, unilateral labeling of motoneurons was achieved when the ipsilateral hindlimb remained attached, suggesting that uptake in motoneurons requires an intact neuromuscular junction. The rostrocaudal incidence and distribution of labeled neurons was uniform in spinal segments L1-L5, with reduced numbers observed in thoracic and L6 spinal segments. Mean total cell labeling was less than 400 per spinal segment, suggesting recruitment from a very small fraction of the neurons contained within the spinal cord (calculated at < 0.1%). These results are consistent with the limited transfer of locomotor-related synaptic activity (Raastad et al. [1996] Neuron 17:729-738) and severe synaptic fatigue (Lev-Tov and Pinco [1992] J Physiol. 447:149-169; Pinco and Lev-Tov [1993] J Neurophysiol. 70:1151-1158; Fleoter and Lev-Tov [1993] J Neurophysiol. 70:2241-2250) observed in the neonatal rat spinal cord.     

Cholecystokinin-like immunoreactivity in the rat spinal cord: Effects of thoracic transection

A study of cholecystokinin-like immunoreactivity in the lumbar (L1-L5) spinal cord segments of rats was realised 24-48 hours after complete thoracic transection (T6-T8). A comparison was made with corresponding spinal cord segments from control and sham-operated animals. The immunocytochemical study with light microscopy showed cholecystokinin-like immunoreactive cell bodies in laminae VII and X at L1-L5, caudal to the transection. In addition, the immunoreactivity was greatly enhanced in bundles of the dorsolateral funiculus compared to sham-operated animals. Our results suggest that part of cholecystokinin-like cell bodies of laminae VII and X send projections to supraspinal sites. Some of these supraspinal projections would go through the dorsolateral funiculus. In the lumbar dorsal horn of operated animals, the immunoreactivity was greatly enhanced in lamina I, while it was slightly decreased in lamina II, compared to control animals. Using electron microscopy, in lamina I, the immunoreactivity localized in different neurites was generally very intense. Moreover, axon terminals showed swelling: their mean size was 0.8-1.8 microns (0.5-1.2 in control animals). This result suggests that some cholecystokinin-like neurons also project to lamina I of rostral cervical segments. In lamina II, numerous degenerating axons were observed (24 hours after thoracic spinal transection). This would suggest that part of descending cholecystokinin-like projections terminate in lamina II.     

Direct agonists for serotonin receptors enhance locomotor function in rats that received neural transplants after neonatal spinal transection

We analyzed whether acute treatment with serotonergic agonists would improve motor function in rats with transected spinal cords (spinal rats) and in rats that received transplants of fetal spinal cord into the transection site (transplant rats). Neonates received midthoracic spinal transections within 48 hr of birth; transplant rats received fetal (embryonic day 14) spinal cord grafts at the time of transection. At 3 weeks, rats began 1-2 months of training in treadmill locomotion. Rats in the transplant group developed better weight-supported stepping than spinal rats. Systemic administration of two directly acting agonists for serotonergic 5-HT(2) receptor subtypes, quipazine and (+/-)-1-[2, 5]-dimethoxy-4-iodophenyl-2-aminopropane), further increased weight-supported stepping in transplant rats. The improvement was dose-dependent and greatest in rats with poor to moderate baseline weight support. In contrast, indirectly acting serotonergic agonists, which block reuptake of 5-HT (sertraline) or release 5-HT and block its reuptake (D-fenfluramine), failed to enhance motor function. Neither direct nor indirect agonists significantly improved locomotion in spinal rats as a group, despite equivalent upregulation of 5-HT(2) receptors in the lumbar ventral horn of lesioned rats with and without transplants. The distribution of immunoreactive serotonergic fibers within and caudal to the transplant did not appear to correspond to restoration of motor function. Our results confirm our previous demonstration that transplants improve motor performance in spinal rats. Additional stimulation with agonists at subtypes of 5-HT receptors produces a beneficial interaction with transplants that further improves motor competence.     

Muscarinic cholinergic receptors in the human spinal cord: differential localization of [H]pirenzepine and [H]quinuclidinylbenzilate binding sites

The localization of muscarinic cholinergic receptor subtypes was studied in the human spinal cord using in vitro labelling of cryostat sections with [³H]quinuclidinylbenzilate (QNB) and [³H]pirenzepine (PZ) followed by autoradiography. The highest densities of [³H]QNB binding were localized in laminae II (substantia gelatinosa) and IX (motor neurons); in contrast, the highest density of [³H]PZ binding was localized to lamina II where the binding density was 22—32% higher than in lamina IX. These results suggest that the M₁ and M₂ muscarinic cholinergic receptor subtypes may be differentially localized in sensory and motor regions of the human spinal cord.     

Altered transcription of glutamatergic and glycinergic receptors in spinal cord dorsal horn following spinal cord transection is minimally affected by passive exercise of the hindlimbs

Gene expression is altered following a spinal transection (STx) in both motor and sensory systems. Exercise has been shown to influence gene expression in both systems post‐STx. Gene expression alterations have also been shown in the dorsal root ganglia and nociceptive laminae of the spinal cord following either an incomplete spinal cord injury (SCI) or a contusive SCI. However, the effect of STx and exercise on gene expression in spinal cord laminae I‐III has not fully been examined. Therefore, the purpose of this study was to determine whether gene expression in laminae I‐III is altered following STx and determine whether superimposed passive exercise of the hindlimbs would influence gene expression post‐STx in laminae I‐III. Laser capture microdissection was used to selectively harvest laminae I‐III of lumbar spinal cord sections, and quantitative RT‐PCR was used to examine relative expression of 23 selected genes in samples collected from control, STx and STx plus exercise rats. We demonstrate that post‐STx, gene expression for metabotropic glutamate receptors 1, 5 and 8 were up‐regulated, whereas ionotropic glutamatergic receptor (Glur2) and glycinergic subunit GLRA1 expression was down‐regulated. Daily exercise attenuated the down‐regulation of Glur2 gene expression in laminae I‐III. Our results demonstrate that in a STx model, gene expression is altered in laminae I‐III and that although passive exercise influences gene expression in both the motor and sensory systems, it had a minimal effect on gene expression in laminae I‐III post‐STx.     

Segmental and laminar organization of the spinal neurons projecting to the periaqueductal gray (PAG) in the cat suggests the existence of at least five separate clusters of spino-PAG neurons

The present retrograde tracing study in the cat describes the spinal cord projections to the periaqueductal gray (PAG), taking into account different regions of the PAG and all spinal segments. Results show that injecting different parts of the PAG leads to different laminar and segmental distributions of labeled spinal neurons. The impression was gained that at least five separate clusters of spinal neurons exist. Cluster I neurons are found in laminae I and V throughout the length of the cord and are probably involved in relaying nociceptive information to the PAG. Cluster II neurons lie in the ventrolateral part of laminae VI-VII of the C1-C4 spinal cord and were labeled by injecting the ventrolateral or lateral part of the rostrocaudal PAG or the deep tectum. Cluster III neurons are located in lamina X of the thoracic and upper lumbar cord and seem to target the PAG and the deep tectum. Cluster IV neurons are located in the medial part of laminae VI-VII of the lumbosacral cord and seem to project predominantly to the lateral and ventrolateral caudal PAG. These neurons may play a role in conveying tactile stimuli to the PAG during mating behavior. Neurons of cluster V are located in the lateral part of lamina I of L6-S2 and in laminae V-VII and X of S1-S3. They are labeled only after injections into the central portion of the lateral and ventrolateral caudal PAG and probably relay information concerning micturition and mating behavior.     

Behavior and binding: correlations between alpha 1-adrenergic stimulation of acoustic startle and alpha 1-adrenoceptor occupancy and number in rat lumbar spinal cord

The relationship between alterations in alpha 1-adrenoceptors and behavioral effects of alpha 1-adrenergic agonists were investigated in a localized region of the rat central nervous system. Direct infusion of the alpha 1-adrenergic agonists, D-amphetamine or phenylephrine. into the subarachnoid space of the lumbar cord (intrathecal administration) increased the amplitude of the acoustic startle reflex, The magnitude of this behavioral facilitation correlated highly with the degree of alpha 1-adrenoceptor occupation measured by [3H]prazosin binding in lumbar spinal tissue. Using an in vitro estimate of receptor occupation, maximal potentiation of startle occurred following approximately 30% occupation of the receptors, using either D-amphetamine or phenylephrine. Intrathecal administration of 6-OHDA produced a 95% decrease in spinal norepinephrine and markedly enhanced the behavioral response to intrathecal phenylephrine as well as the number of alpha 1-adrenoceptors. The correlation between the time course of the behavioral and binding changes was 0.99. No change in receptor affinity (KD) or receptor occupation by phenylephrine was found after 6-OHDA. The data indicate that receptor binding parameters do have predictive value for behavior, especially if localized regions of the nervous system, critical to the behavior, are analyzed.     

Receptive fields and responses to ionophoretically applied noradrenaline and 5-hydroxytryptamine of units recorded in laminae I-III of cat dorsal horn

Recordings were made with carbon fibre microelectrodes from 87 units in laminae I, II and III of the spinal cord in anaesthetized cats, and responses of the units to ionophoretically applied noradrenaline (NA) and 5-hydroxytryptamine (5-HT) were observed. Units with low threshold receptive fields were situated in laminae II and III, while those with high threshold or wide dynamic range fields were mainly restricted to laminae I and II. NA excited nearly half of the units in laminae I and II but had no effect on most units in lamina III. 5-HT excited 68% of the units and these were distributed throughout all 3 laminae. Excitations, particularly by 5-HT, could be very prolonged. NA and 5-HT excited units in all 3 receptive field classes. A few units in lamina I were inhibited. It is suggested that the cells recorded from in the present experiments may be inhibitory interneurones which act on large dorsal horn cells or on their primary afferent inputs.     

Effects of 5,7-dihydroxytryptamine on serotonin1 and serotonin2 receptors throughout the rat central nervous system using quantitative autoradiography

The effects of the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), on serotonin1 (5-HT1) and 5-HT2 receptors were investigated using the high degree of resolution provided by quantitative autoradiography in an effort to determine the synaptic location of these receptors. 5,7-DHT treatment resulted in a decrease in 5-HT1 binding in the dentate gyrus and CA3c/4 of the anterior hippocampus and in the dorsal raphe nucleus, whereas no changes were observed in the posterior hippocampus nor in many other brain structures. 5-HT2 receptors exhibited no changes in any brain area examined in response to 5,7-DHT treatment, despite over 90% serotonin depletion in most of the forebrain nuclei examined. The results indicate that at least some of the 5-HT1 sites labelled by [3H]5-HT in the hippocampus and dorsal raphe nucleus are presynaptic, whereas 5-HT2 receptors are probably postsynaptic. In addition, the distribution profiles of 5-HT1 and 5-HT2 binding sites were compared in the rat central nervous system at various anatomical levels. 5-HT1 binding sites were identified using [3H]5-HT, while 5-HT2 binding sites were labelled with [3H]ketanserin. Both receptor subtypes displayed distinctly different localization patterns, which, in most cases was the inverse of the other pattern. In the brainstem it is significant that 5-HT2 receptors are concentrated in the facial nucleus and the motor nucleus of the trigeminal nerve, areas known to influence head and facial movement. The serotonin-mediated head-shake response occurs when 5-HT2 receptors are activated. In contrast, 5-HT1 receptors are distributed throughout the brainstem and in specific portions of the spinal cord. These areas are thought to control the serotonin behavioral syndrome and this behavior is 5-HT1A-mediated. All raphe nuclei were devoid of 5-HT2 receptors; only 5-HT1 receptor were found in these nuclei. Correlations with serotonin terminal distribution patterns are discussed. The pattern of 5-HT2 receptor distribution was also compared with the pattern of alpha 1 receptors, using [3H]prazosin in order to determine whether [3H]ketanserin significantly labels alpha 1 receptors. Although some similarities exist, overlap of binding did not occur in other nuclei, indicating that alpha 1 contamination of this system is probably negligible.     

Identified spinal motoneurons of young rats possess nicotinic acetylcholine receptors of the heteromeric family

The aim of the present study was to determine whether, in young rats, spinal motoneurons possess functional nicotinic acetylcholine receptors. Motoneurons were identified either by retrograde labelling or by choline acetyltransferase immunohistochemistry. Whole-cell recordings were performed in spinal cord slices cut at the lumbar level. In voltage clamp, acetylcholine evoked a rapidly activating inward current. In current clamp, it depolarized the motoneuron membrane and induced action potential firing. The acetylcholine-evoked current was strongly reduced by d-tubocurarine or dihydro-beta-erythroidine, broad spectrum nicotinic antagonists, but was almost insensitive to methyllycaconitine, a nicotinic antagonist selective for receptors containing the alpha7 subunit. Moreover, exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane, an alpha7-specific agonist, was without effect. In young animals, light-microscopic autoradiography showed that in the central grey matter all laminae were intensely and equally labelled by [3H]epibatidine. A dense [125I]-alpha-bungarotoxin binding was also found in all laminae, with slightly lower levels in the superficial layers of the dorsal horns and in the ventral part of the grey matter. In adults, the density of [3H]epibatidine binding sites was much lower in the entire grey matter, except in layer 2 of the dorsal horn, and [125I]-alpha-bungarotoxin binding sites were present only in some selected areas. Our data indicate that spinal motoneurons possess functional nicotinic receptors of the heteromeric type and suggest that nicotinic cholinergic transmission may play a significant role in the developing spinal cord.     

Ovarian steroid modulation of [H]muscimol binding in the spinal cord of the rat

[³H]Muscimol binding was measured in the lumbar spinal cord of female rats by in vitro quantitative autoradiography. Ovariectomized rats were treated subcutaneously with either oil, estradiol benzoate (EB) or EB plus progesterone (P) in a regime known to reliably induce sexual receptivity. The level of [³H]muscimol binding was highest in laminae I–III and in the region around the central canal. Binding was lower in laminae IV–VI and was frequently undetectable in the ventral horn. There was a significant increase in the level of binding in laminae I–III after EB treatment. There was also a significant increase after treatment with EB+P in comparison to both the ovariectomized and EB-treated groups in this same region of the spinal cord.