Corticotropin releasing factor-like immunoreactivity in afferent projections to the sacral spinal cord of the cat

Distribution and origin of corticotropin releasing factor (CRF) in the thoraco-lumbar and sacral spinal cord of the cat has been studied using immunohistochemical method. CRF immunoreactive (CRF-IR) nerve fibers and terminals were most prominent in dorsal part of sacral spinal cord. In the sacral segments of the spinal cord, immunoreactivity for CRF was detected in a prominent bundle of axons and varicosities extending from Lissauer's tract (LT) along the lateral edge of the superficial dorsal horn (laminae I and II) to laminae V at the base of the dorsal horn. Individual CRF-IR fibers passed from the bundle in ventral medial and ventrolateral directions to the dorsal commissure and the sacral preganglionic nucleus (SPN), respectively. The bundle of CRF-IR axons closely resembled vasoactive intestinal polypeptide (VIP) containing fibers in LT and on the lateral edge of the dorsal horn. Sacral dorsal root transection eliminated both the CRF and VIP fiber staining in the dorsal horn. Spinal transection at the T12-T13 segmental level did not influence the CRF- or VIP-IR. Less intense CRF-IR was also present in fibers in: (1) the dorsal lateral funiculus adjacent to LT, (2) the superficial layers of the dorsal horn and intermediolateral nucleus at thoracolumbar spinal levels, (3) the ventral horn, including Onuf's nucleus, (4) the intermediate gray matter including the dorsal gray commissure, and (5) the SPN. The similarity in the distribution of CRF-IR and pelvic nerve afferent projections in the sacral spinal cord raises the possibility that CRF may be a transmitter in afferent neurons innervating the pelvic viscera.     

Quantitative analysis of substance P-immunoreactive boutons on physiologically characterized dorsal horn neurons in the cat lumbar spinal cord

A quantitative analysis of substance P (SP)-immunoreactive (IR) terminals contacting physiologically characterized dorsal horn neurons was performed. Three types of neuron were studied: nociceptive specific (NS) from lamina I (n = 3), wide dynamic range (WDR) from laminae II-IV (n = 3), and nonnociceptive (NN) from lamina IV (n = 3). The nociceptive response of focus was a slow, prolonged depolarization to noxious stimuli, because this response was previously shown to be blocked by selective neurokinin-1 (NK-1) receptor antagonists. Ultrastructural immunocytochemistry was used to quantify the relative number of SP-IR boutons apposed to the intracellularly labeled cell per unit of length (density). Densities of the total population (SP immunoreactive+nonimmunoreactive) of apposed boutons were similar in all three regions (cell body, proximal and distal dendrites) for the three functional types of neuron. NS neurons received a significantly higher density of appositions from SP-IR boutons than NN cells in all three regions. However, compared to WDR cells, NS cells possessed a significantly higher density of appositions from SP-IR boutons only in the cell body and proximal dendrites. WDR cells had a higher density of appositions from SP-IR boutons than NN cells, but only in the proximal and distal dendrites. On average, 33.5% of the SP-IR boutons apposed to the cells displayed a synaptic contact. Finally, 30-45% of the SP-IR boutons apposed to the cells colocalized calcitonin gene-related protein (CGRP) immunoreactivity, indicating their primary sensory origin. The data indicate a direct correlation between the amount of SP-IR input and the nociceptive nature of the cells and suggest that SP acts on NK-1 receptors at a short distance from its release site.     

Selective cortical and segmental control of primary afferent depolarization of single muscle afferents in the cat spinal cord

This study was primarily aimed at investigating the selectivity of the cortico-spinal actions exerted on the pathways mediating primary afferent depolarization (PAD) of muscle spindle and tendon organ afferents ending within the intermediate nucleus at the L6-L7 segmental level. To this end we analyzed, in the anesthetized cat, the effects produced by electrical stimulation of sensory nerves and of the cerebral cortex on (a) the intraspinal threshold of pairs of single group I afferent fibers belonging to the same or to different hindlimb muscles and (b) the intraspinal threshold of two collaterals of the same muscle afferent fiber. Afferent fibers were classified in three categories, according to the effects produced by stimulation of segmental nerves and of the cerebral cortex. Twenty-five of 40 fibers (62.5%) were depolarized by stimulation of group I posterior biceps and semitendinosus (PBSt) or tibialis (Tib) fibers, but not by stimulation of the cerebral cortex or of cutaneous and joint nerves, which instead inhibited the PBSt- or Tib-induced PAD (type A PAD pattern, usually seen in Ia fibers). The remaining 15 fibers (37.5%) were all depolarized by stimulation of the PBSt or Tib nerves and the cerebral cortex. Stimulation of cutaneous and joint nerves produced PAD in 10 of those 15 fibers (type B PAD pattern) and inhibited the PBSt- or Tib-induced PAD in the 5 remaining fibers (type C PAD pattern). Fibers with a type B or C PAD pattern are likely to be Ib. Not all sites in the cerebral cortex inhibited with the same effectiveness the segmentally induced PAD of group I fibers with a type A PAD pattern. With the weakest stimulation of the cortical surface, the most effective sites that inhibited the PAD of individual fibers were surrounded by less effective sites, scattered all along the motor cortex (area 4gamma and 6) and sensory cortex (areas 3, 2 and 1), far beyond the area of projection of group I fibers from the hindlimb. With higher strengths of cortical stimulation, the magnitude of the inhibition was also increased, and previously ineffective or weakly effective sites became more effective. Maps obtained when using the weakest cortical stimuli have indicated that the most effective regions that produced PAD of group I fibers with a type B or type C PAD pattern were also scattered throughout the sensory-motor cortex, in the same general area as those that inhibited the PAD of group I afferents with a type A PAD pattern. In eight fibers with a type A PAD pattern it was possible to examine the intraspinal threshold of two collaterals of the same single afferent fiber ending within the intermediate nucleus at the L7 segmental level. In six fibers, stimulation of the PBSt nerve with trains of pulses between 1.5 and 1.86 times threshold (xT) produced a larger PAD in one collateral than in the other. In seven fibers, stimulation of the sensory-motor cortex and of cutaneous nerves produced a larger inhibition of the PBSt-induced PAD in one collateral than in the other. The ratio of the cortically induced inhibition of the PAD elicited in the two collaterals could be modified by changing the strength of cortical and of PBSt stimulation. In three fibers it was possible to inhibit almost completely the background PAD elicited in one collateral while having little or no effect on the PAD in the other collateral. Changes in the intraspinal threshold of pairs of collaterals following electrical stimulation of segmental nerves and of the somato-sensory cortex were examined in three fibers with a type B and two fibers with a type C PAD pattern. In four fibers the PAD elicited by stimulation of cutaneous (4-20xT) and muscle nerves (1.54-3.7xT), or by stimulation of the sensory-motor cortex, was of different magnitude in the two collaterals. In two experiments it was possible to find cortical sites in which weak surface stimulation produced PAD in one collateral only. (ABSTRACT TRUNCATED)     

Effects of caudal traction of the spinal cord on evoked spinal cord potentials in the cat

This study attempts clarify the mechanism of neurological deficits in tethered cord syndrome using evoked spinal cord potentials (ESCPs). ESCPs in response to both sciatic nerve (SN-ESCP) and spinal cord stimulation (SC-DESCP) were recorded from the dorsal epidural space. With a fixed degree of caudal traction on the spinal cord in ten cats for 2-4 hours, ESCPs were increased in amplitude in the N1 and N2 deflections of the SC-DESCPs to 158% and 154% at L5 and decreased to 91% and 76% after transient augmentation at L3. On the other hand, the amplitude in the N1 deflection of the SN-ESCPs at L3 and L5 was decreased to 40% and 68%. These findings suggest that not only the force but also the duration of traction influence the degree of the spinal cord dysfunction. When the spinal cords of 17 cats received compression with traction and without traction, the SN-ESCPs of the former became positive earlier than that of the latter. The extent of the recovery in amplitude of both SC-DESCPs and SN-ESCPs propagated over compression site was far limited in the former than in the latter. These results would indicate that the spinal cord subjected to traction is vulnerable to compression.     

Nerve growth factor receptor-like immunoreactivity in the human spinal cord

Nerve growth factor (NGF) receptor-like immunoreactivity has been demonstrated in the normal human adult spinal cord using the monoclonal antibody ME20.4. Intense immunoreactivity was associated with fibres and terminals in the substantia gelatinosa. In lamina IX the neuropil demonstrated punctate staining, the motor neurons themselves being negative. At thoracic levels occasional neurons of the intermediolateral column cell group were NGF receptor positive. Fine axonal and punctate terminal reactivity was observed in the gracile fasciculus, corresponding to axons in transverse section. Similar, though slightly less dense immunoreactivity was observed in the cuneate fasciculus. The demonstration of NGF receptor immunoreactivity may provide a useful marker of sensory innervation in the human spinal cord.     

Reconstruction of trajectory of primary afferent collaterals in the dorsal horn of the cat spinal cord, using Golgi-stained serial sections

Using Golgi-stained serial sections obtained at the sacro-caudal levels of the cat spinal cord, it was possible to reconstruct the trajectory of primary afferents. They were classified into two groups: reliable primary afferents directly traced from the dorsal root and probable primary afferents traced from the dorsal funiculus or Lissauer's tract. The diameters of the reliable primary afferents vary from 0.88-1.88 mum. According to their courses, reliable primary afferents as well as probable primary afferents were classified into three groups: the first is distributed to both medial and lateral halves of the dorsal horn, the second to the medial half, and the third to the lateral half. Commissural fibers were also observed among the probable primary afferents. The rostro-caudal and medio-lateral extents of reliable primary afferents are found to be between 250 and 950 mum and 270 and 700 mum respectively, while those of the probable primary afferents were between 125 and 670 mum and 270 and 1,640 mum respectively. These primary afferent fibers are connected with at least two or more laminae of the dorsal horn gray matter.     

Compression of spinal cord caused by spinal deformation in a cat

An 8-month-old cat with bilateral hindlimb paresis was admitted. Radiography revealed a curvature of the thoracic spine and a partial deformation of thoracolumbar vertebrae (dorsal extrusion of the cranial and caudal edges). Compression of the spinal cord (T9-L1 and L6-7) was found by myelography. Medicinal treatment did not yield the desired result. Hemilaminectomy and laminectomy were done to relieve pressure on the spinal cord leading to improved gait. It is, however, not clear whether this syndrome was primarily caused by metabolic or genetic factors.     

Topographical organization of group II afferent input in the rat spinal cord

Recent data suggest that patients with Alzheimer's disease (AD) are able to show perceptual priming and, to some extent, conceptual priming for material which has preexisting representations in memory, and that normal elderly subjects are able to automatically activate pre-existing representations in both perceptual and conceptual priming tasks. An important question concerns the capacity of showing priming for materials without pre-existing representations in memory in normal and pathological aging. In order to address this issue, 20 patients with mild AD, 20 elderly controls and 20 young controls subjects were assessed with a paradigm of priming for new verbal associations. Neither the patients nor the normal elderly subjects demonstrated priming effects for new associations, while young subjects showed significant priming effects. These results suggest that the absence of priming for new verbal associations is attributable more to an effect of aging than to a specific effect of Alzheimer's disease.     

Sprouting of primary afferent fibers after spinal cord transection in the rat

After spinal cord injury, hyper-reflexia can lead to episodic hypertension, muscle spasticity and urinary bladder dyssynergia. This condition may be caused by primary afferent fiber sprouting providing new input to partially denervated spinal interneurons, autonomic neurons and motor neurons. However, conflicting reports concerning afferent neurite sprouting after cord injury do not provide adequate information to associate sprouting with hyper-reflexia. Therefore, we studied the effect of mid-thoracic spinal cord transection on central projections of sensory neurons, quantified by area measurements. The area of myelinated afferent arbors, immunolabeled by cholera toxin B, was greater in laminae I-V in lumbar, but not thoracic cord, by one week after cord transection. Changes in small sensory neurons and their unmyelinated fibers, immunolabeled for calcitonin gene-related peptide, were assessed in the cord and in dorsal root ganglia. The area of calcitonin gene-related peptide-immunoreactive fibers in laminae III-V increased in all cord segments at two weeks after cord transection, but not at one week. Numbers of sensory neurons immunoreactive for calcitonin gene-related peptide were unchanged, suggesting that the increased area of immunoreactivity reflected sprouting rather than peptide up-regulation. Immunoreactive fibers in the lateral horn increased only above the lesion and in lumbar segments at two weeks after cord transection. They were not continuous with dorsal horn fibers, suggesting that they were not primary afferent fibers. Using the fluorescent tracer DiI to label afferent fibers, an increase in area could be seen in Clarke's nucleus caudal to the injury two weeks after transection. In conclusion, site- and time-dependent sprouting of myelinated and unmyelinated primary afferent fibers, and possibly interneurons, occurred after spinal cord transection. Afferent fiber sprouting did not reach autonomic or motor neurons directly, but may cause hyper-reflexia by increasing inputs to interneurons.     

Ascending pressor and depressor pathways in the cat spinal cord

Blood pressure responses to stimulation of the cervical dorsolateral sulcus (DLS) of the spinal cord and lumbar dorsal roots were studied in anesthetized, vagotomized, and paralyzed cats. Stimulation of the lumbar dorsal roots elicited pressor responses with high-frequency stimulation (50 Hz, 10 V, 1 ms) and depressor responses with low-grequency stimulation (1 Hz, 10 V, 1 ms). Pressor responses were converted to depressor responses after bilateral lesions were made in the DLS area rostral to the site of stimulation. These results suggest that the ascending spinal pressor pathways are localized in the DLS region. Furthermore, these depressor responses were abolished by placing additional bilateral lesions in the dorsolateral funiculus (DLF) rostral to the site of stimulation. These data indicate the presence of ascending depressor pathways in the DLF which are anatomically separate from pressor pathways. Both pathways were found to be bilateral systems, and decussation of fibers appears to be complete within three segments rostral to their entry into the spinal cord. Ascending depressor pathways have a low optimal frequency of activation, as opposed to the ascending pressor pathways which have a relatively high optimal frequency of activation. Neurophysiological evidence obtained by recording unit activity from the cervical sympathetic trunk confirmed the localization of the ascending pressor and depressor pathways.     

Astroglial distribution of neurokinin-2 receptor immunoreactivity in the rat spinal cord

Two mouse monoclonal antibodies, 11H9.1 and 1G7.10, raised against the COOH-terminus peptide (359-390) of the rat neurokinin-2 receptor, were used to visualize by light and electron microscope immunocytochemistry the distribution of this receptor in adult rat spinal cord. At all spinal levels, immunoreactivity was mainly observed in two narrow crescentic zones bordering the gray matter of the dorsal and ventral horns, and around the central canal. In the light microscope, this labelling was the densest within the outer part of lamina I facing the dorsal column, where it took the form of minute dots and streaks scattered in the neuropil. In the electron microscope, such a localization was exclusively astrocytic and essentially involved astrocytic leaflets, as indicated by the size and irregular shape of the immunostained processes, their location between and around neuronal profiles, and their occasional display of glial filaments. The diaminobenzidine reaction product showed some predilection for the plasma membrane and was occasionally seen at gap junctions of these labelled processes. Many labelled astrocytic leaflets were observed in the immediate vicinity of axon terminals containing large dense-cored vesicles, and around fibres morphologically identifiable as primary afferent, unmyelinated C-fibres. These observations suggest that astrocytic neurokinin-2 receptors could define the effective sphere of neurokinin A neuromodulation in rat spinal cord, via alterations in the regulation of the extracellular environment and glutamate uptake by astrocytes and/or the release of putative astroglial mediators. The astrocyte neurokinin-2 receptors, activated by extrasynaptic neurokinin A, might thus co-operate with neurokinin-1 and neurokinin-3 neuronal receptors in the modulation of nociceptive information.     

Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord

Substance P plays an important role in the transmission of pain-related information in the dorsal horn of the spinal cord. Recent immunocytochemical studies have shown a mismatch between the distribution of substance P and its receptor in the superficial laminae of the dorsal horn. Because such a mismatch was not observed by using classical radioligand binding studies, we decided to investigate further the issue of the relationship between substance P and its receptor by using an antibody raised against a portion of the carboxyl terminal of the neurokinin 1 receptor and a bispecific monoclonal antibodies against substance P and horseradish peroxidase. Light microscopy revealed a good correlation between the distributions of substance P and the neurokinin 1 receptor, both being localized with highest densities in lamina I and outer lamina II of the spinal dorsal horn. An ultrastructural double-labeling study, combining preembedding immunogold with enzyme-based immunocytochemistry, showed that most neurokinin 1 receptor immunoreactive dendrites were apposed by substance P containing boutons. A detailed quantitative analysis revealed that neurokinin 1 receptor immunoreactive dendrites received more appositions and synapses from substance P immunoreactive terminals than those not expressing the neurokinin 1 receptor. Such preferential innervation by substance P occurred in all superficial dorsal horn laminae even though neurokinin 1 receptor immunoreactive dendrites were a minority of the total number of dendritic profiles in the above laminae. These results suggest that, contrary to the belief that neuropeptides act in a diffuse manner at a considerable distance from their sites of release, substance P should act on profiles expressing the neurokinin 1 receptor at a short distance from its site of release.     

Penile erection produced by microstimulation of the sacral spinal cord of the cat

The homeobox gene goosecoid is expressed in the Spemann organizer tissue of gastrulating vertebrate embryos, and in the craniofacial region and appendicular skeleton during organogenesis. The goosecoid knockout mutant mouse revealed defects related to the second phase of expression. Here we describe new expression sites in the developing trachea and external genitalia, and in the developing shoulder and hip joint with their associated ligaments and muscles. We show that mutant mice display abnormalities in the forming trachea and appendicular skeleton related to these sites of gene expression. In addition, we discuss evidence for the existence of at least three goosecoid genes in vertebrates, which may account for the lack of a defect of axial patterning in goosecoid mutant mice.     

Distinct patterns of NCAM expression are associated with defined stages of murine hair follicle morphogenesis and regression

Hair follicle development, growth (anagen), and regression (catagen) largely result from bidirectional epithelial-mesenchymal interactions whose molecular basis is still unclear. Because adhesion molecules are critically involved in pattern formation and because the fundamental importance of neural cell adhesion molecule (NCAM) for feather development has been demonstrated, we studied the protein expression patterns of NCAM during hair follicle development and regression in the C57BL/6 mouse model. During murine hair follicle development, NCAM immunoreactivity (IR) was first detected on epithelial hair placodes and later on selected keratinocytes in the distal outer root sheath. Mesenchymal NCAM immunoreactivity (IR) was noted on fibroblasts of the future dermal papilla (DP) and the perifollicular connective tissue sheath. Fetal hair follicle elongation coincided with strong, ubiquitous dermal NCAM IR, which remained strong until the follicles entered into their first neonatal catagen. At this time, the strong interfollicular dermal NCAM IR decreased substantially. During consecutive hair cycles, mesenchymal NCAM IR was seen exclusively on DP and perifollicular connective tissue sheath fibroblasts and on the trailing cells of regressing catagen hair follicles. These highly restricted and developmentally controlled expression patterns suggest an important role for NCAM in hair follicle topobiology during morphogenesis and cyclic remodeling of this miniorgan.     

The mechanism of inhibition of neuronal activity by opiates in the spinal cord of cat

Extra- and intracellular recordings from motoneurones, interneurones and dorsal horn neurones (laminae 4 and 5) were obtained from the lumbar segments (L6-L7) of spinalised (Th 9/10) or pentobarbital-anaesthetised and anaemically decorticated cats. In the majority of spinal neurones microelectrophoretically applied morphine and levorphanol reversibly depressed spontaneous as well as stimulus-evoked and L-glutamate- or acetylcholine-induced activity. There is evidence that opiates block L-glutamate-induced depolarisations by impairing the Na+-influx triggered at the postsynaptic membrane. These depressant effects of opiates could be antagonised by naloxone, and, except in a few cases, were not associated with hyperpolarisation of the cell. Dextrorphan, the D+ enantiomer of levorphanol, displayed no such depressant actions, indicating that stereospecific receptors mediate the depressant effects of opiates. Phoretically applied atropine, procaine and Ca2+ ions have anti-glutamate and anti-acetylcholine actions similar to opiates, but these actions were not antagonised by naloxone. The hyperpolarising effect of glycine was not influenced at dose levels of opiates sufficient to suppress depolarisation induced by L-glutamate or acetylcholine. Microelectrophoretically administered morphine and levorphanol slowed the rate of rise of mono- and polysynaptic EPSPs by a naloxone-antagonisable mechanism at dose levels where almost no alteration in spike shape was detectable. Increased doses of morphine and levorphanol reduced the amplitude of IPSPs and completely blocked or reduced the amplitude of both direct- and antidromically-evoked spikes. These effects of increased doses of opiates were not antagonised by naloxone. Intravenous injection of 2 mg/kg of morphine or 20 mug/kg of Fentanyl mimicked the suppression of spontaneous and evoked neuronal activity observed after phoretic administration. This depressant action of systemically applied opiates could be transiently antagonised by phoretic administration of naloxone. The results are discussed with respect to a stereospecific action of opiates at a postsynaptic receptive site in the spinal cord.     

Estrogen receptor-immunoreactive neurons are present in the female rat lumbosacral spinal cord

We investigated the prevalence of quality of care deficiencies in 4,324 Medicare-reimbursed episodes of care provided by 47 home health agencies. The quality of care protocol consisted of a process-oriented, systematic record review by a trained nurse reviewer. Results suggest that an estimated 14.4% of home health care episodes had quality deficiencies with the potential for or actual adverse effects on the patient. Multivariate analyses revealed that the complexity of patients' needs increased the likelihood and severity of the quality problems. Agency ownership was not related to risk of a quality problem, but regional variation in agency effects was observed. Specific problem areas were identified that suggested several ways that home health care could be improved.     

Mechanosensitive afferent units in the hypogastric nerve of the cat

1. Recordings have been made from ninety single afferent units in the hypogastric and lumbar splanchnic nerves of the cat. 2. The majority of units examined had properties similar to those previously described in the splanchnic nerve: they were slowly adapting mechanoreceptors with one to six punctate mechanosensitive sites distributed mainly along blood vessels as they approached the viscera in peritoneal ligaments; they had a wide range of mechanical thresholds and conduction velocities in the range 0.5-24 m s-1. 3. Receptive fields were found over the bladder base or its peritoneal ligaments or both, on the uterus or broad ligament or both, on the colon or mesocolon or both, and in association with the ureter, vas deferens, prostate or pelvic fat pads. 4. Discharges from afferent units associated with the bladder were investigated during spontaneous (or reflex) bladder contractions, passive distensions, and tetanic contractions induced by electrical stimulation of the sacral spinal cord. The mean spike rates of the adapted (tonic) discharges, observed during distensions and induced tetani, differed over part of the range of intravesical pressures examined. Their behaviour is discussed in relation to the concept of 'in series' tension receptors within the bladder wall.     

Increases in protein kinase C gamma immunoreactivity in the spinal cord of rats associated with tolerance to the analgesic effects of morphine

Our previous studies have indicated a critical role of protein kinase C (PKC) in intracellular mechanisms of tolerance to morphine analgesia. In the present experiments, we examined (1) the cellular distribution of a PKC isoform (PKC gamma) in the spinal cord dorsal horn of rats associated with morphine tolerance by utilizing an immunocytochemical method and (2) the effects of the N-methyl-D-aspartate receptor antagonist MK-801 on tolerance-associated PKC gamma changes. In association with the development of tolerance to morphine analgesia induced by once daily intrathecal administration of 10 micrograms morphine for eight days, PKC gamma immunoreactivity was clearly increased in the spinal cord dorsal horn of these same rats. Within the spinal cord dorsal horn of morphine tolerant rats, there were significantly more PKC gamma immunostained neurons in laminae I-II than in laminae III-IV and V-VI. Such PKC gamma immunostaining was observed primarily in neuronal somata indicating a postsynaptic site of PKC gamma increases. Moreover, both the development of morphine tolerance and the increase in PKC gamma immunoreactivity were prevented by co-administration of morphine with 10 nmol MK-801 between Day 2 and Day 7 of the eight day treatment schedule. In contrast, PKC gamma immunoreactivity was not increased in rats receiving a single i.t. administration of 10 micrograms morphine on Day 8, nor did repeated treatment with 10 nmol MK-801 alone change baseline levels of PKC gamma immunoreactivity. These results provide further evidence for the involvement of PKC in NMDA receptor-mediated mechanisms of morphine tolerance.