Localization of glutamate receptors in dorsal horn of rat spinal cord

Immunocytochemical localization of metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (NMDA-type: NMDAR1 and NMDAR2A-C; AMPA-type: GluR1-4) was performed on sections of rat dorsal horn. Immunoreactivity for mGluR1 alpha was detected in laminae I-III of the dorsal horn, whilst mGluR2/3 immunoreactivity was detected primarily in lamina III. Immunoreactivity for NMDAR1, GluR1, GluR2, GluR2/3, GluR4 and GluR5/6/7 was strongly localized in neuronal elements of laminae I-III. Immunoreactivity for NMDAR2B was localized in laminae I-III. No mGluR5, NMDAR2A and NMDAR2C immunoreactivity was detected. In addition, immunoreactivity for receptors was found to co-localize with immunoreactivity for glutamate in the dorsal horn. The present results indicate that glutamate receptors are differentially localized in neuronal elements of dorsal horn where receptor-neurotransmitter interaction takes place.     

A novel metabotropic glutamate receptor agonist: marked depression of monosynaptic excitation in the newborn rat isolated spinal cord

1. Neuropharmacological actions of a novel metabotropic glutamate receptor agonist, (2S,1'R,2'R,3'R)-2(2,3-dicarboxycyclopropyl)glycine (DCG-IV), were examined in the isolated spinal cord of the newborn rat, and compared with those of the established agonists of (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD). 2. At concentrations higher than 10 microM, DCG-IV caused a depolarization which was completely blocked by selective N-methyl-D-aspartate (NMDA) antagonists. The depolarization was pharmacologically quite different from that caused by L-CCG-I and (1S,3R)-ACPD. 3. DCG-IV reduced the monosynaptic excitation of motoneurones rather than polysynaptic discharges in the nanomolar range without causing postsynaptic depolarization of motoneurones. DCG-IV was more effective than L-CCG-I, (1S,3R)-ACPD or L-2-amino-4-phosphonobutanoic acid (L-AP4) in reducing the monosynaptic excitation of motoneurones. 4. DCG-IV (30 nM-1 microM) did not depress the depolarization induced by known excitatory amino acids in the newborn rat motoneurones, but depressed the baseline fluctuation of the potential derived from ventral roots. Therefore, DCG-IV seems to reduce preferentially transmitter release from primary afferent nerve terminals. 5. Depression of monosynaptic excitation caused by DCG-IV was not affected by any known pharmacological agents, including 2-amino-3-phosphonopropanoic acid (AP3), diazepam, 2-hydroxysaclofen, picrotoxin and strychnine. 6. DCG-IV has the potential of providing further useful information on the physiological function of metabotropic glutamate receptors.     

Characterization of commissural interneurons in the lumbar region of the neonatal rat spinal cord

Neurons with axons that extend to the contralateral side of the spinal cord--commissural interneurons (CINs)--coordinate left/right alternation during locomotion. Little is known about the organization of CINs in the mammalian spinal cord. To determine the numbers, distribution, dendritic morphologies, axonal trajectories, and termination patterns of CINs located in the lumbar spinal cord of the neonatal rat, several different retrograde and anterograde axonal tracing paradigms were performed with fluorescent dextran amines and the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). CINs with ascending (aCINs) and descending (dCINs) axons were labeled independently. The aCINs and dCINs occupied different but overlapping domains within the transverse plane. The aCINs were clustered into four recognizable groups, and the dCINs were clustered into two recognizable groups. All dCINs and most aCINs were located within the gray matter, with somata ranging from 10-30 microm in diameter and with large, multipolar dendritic trees. One group of aCINs was located outside the gray matter along the dorsal and dorsolateral margin and had dendrites that were nearly confined to the dorsolateral surface. All CIN axons traversed the ventral commissure at right angles to the midline. CIN axons coursed up to six or seven segments rostrally and/or caudally in the ventral and ventrolateral white matter and gave off collaterals over a shorter range, predominantly to the ventral gray matter. These findings show that the lumbar spinal cord of the neonatal rat contains substantial numbers of CINs with axon projections and collateral ranges spanning several segments and that CINs projecting rostrally vs. caudally have different distributions in the transverse plane. The study provides an anatomical framework for future electrophysiological studies of the spinal neuronal circuits underlying locomotion in mammals.     

Interactions between metabotropic and ionotropic glutamate receptor agonists in the rat spinal cord in vivo

Microelectrophoretic application of the non-selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] and the group I selective mGluR agonist (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG] potentiated the responses of rat spinal neurones to the cyclically-ejected ionotropic excitatory amino acid (EAA) agonists NMDA, AMPA and kainate in vivo. Potentiation was not selective between the three ionotropic responses and was paralleled by an enhancement of background activity in spontaneously active cells. "Correcting" spike count data for this increase in background activity showed that the EAA responses were not potentiated beyond the apparent enhancement of cell excitability. Neither mGluR agonist produced potentiation when NMDA/AMPA cycling was superimposed on background discharge held constant with kainate. It is concluded that potentiation produced by both (1S,3R)-ACPD and (RS)-3,5-DHPG is secondary to an enhancement of cell excitability rather than being due to a specific interaction with ionotropic EAA receptors. The mechanism of excitability enhancement cannot be determined by extracellular recording, but group I mGluRs are most likely to be responsible.     

On the release of glutamate and aspartate in the basal ganglia of the rat: interactions with monoamines and neuropeptides

Experiments were designed to determine the in vivo cell-cycle phase of lymphocytes in secondary lymphoid follicles, and whether B-cell proliferation and apoptosis occur within follicular dendritic cell (FDC)-associated clusters. Using frozen serial sections of human tonsils, lymphoid follicles were stained to reveal histone H3 mRNA, as an S-phase marker, using in situ hybridization, and stained immunohistochemically with antibodies against cyclin E as a late G1 phase marker, cyclin B1 and p34cdc2 as S-G2-M phase markers, and Ki-67 as a marker of cycling cells. Each LF was divided into five zones: mantle zone, outer zone, apical light zone, basal light zone and dark zone, with the help of haematoxylin and eosin staining, and a CD23 immunostain. The rate of occurrence of positively labelled cells was calculated by dividing the number of positive cells by the number of all cells in each zone. The cells that were positive for cyclin E, histone H3 mRNA, cyclin B1, p34cdc2, and Ki-67 were found most frequently in the dark zone (54.5 +/- 6.6%, 22.0 +/- 5.7%, 36.7 +/- 14.5%, 40.0 +/- 10.2%, and 59.0 +/- 13.4%, respectively), followed by the outer zone (52.7 +/- 7.8%, 14.9 +/- 4.1%, 22.9 +/- 9.7%, 24.9 +/- 7.9%, and 44.6 +/- 12.3%, respectively), showing that both the outer zone and the dark zone contain many proliferating lymphocytes. Furthermore, FDC-associated clusters and free lymphocytes were obtained from enucleated germinal centres, using enzymatic digestion. The rates of occurrence of cells that were positive for cyclin B1 and Ki-67 within the clusters (7.2 +/- 1.9% and 37.9 +/- 10.5% respectively) were significantly lower than those of free lymphocytes outside the clusters (22.2 +/- 4.0% and 62.8 +/- 14.0%, respectively). The rates of occurrence of apoptotic bodies and cells within the clusters, as detected by in situ tailing or in situ nick translation (0.2 +/- 0.4% and 0.4 +/- 0.4%, respectively) were significantly lower than those outside the clusters (1.1 +/- 0.3, 1.6 +/- 0.5%, respectively). These results suggest that FDC-associated clusters are not the site of proliferation, and that they rarely contain apoptotic bodies and cells of B lymphocytes.     

Contribution of NMDA and non-NMDA glutamate receptors to locomotor pattern generation in the neonatal rat spinal cord

The motor programme executed by the spinal cord to generate locomotion involves glutamate-mediated excitatory synaptic transmission. Using the neonatal rat spinal cord as an in vitro model in which the locomotor pattern was evoked by 5-hydroxytryptamine (5-HT), we investigated the role of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in the generation of locomotor patterns recorded electrophysiologically from pairs of ventral roots. In a control solution, 5-HT (2.5-30 microM) elicited persistent alternating activity in left and right lumbar ventral roots. Increasing 5-HT concentration within this range resulted in increased cycle frequency (on average from 8 to 20 cycles min-1). In the presence of NMDA receptor antagonism, persistent alternating activity was still observed as long as 5-HT doses were increased (range 20-40 microM), even if locomotor pattern frequency was lower than in the control solution. In the presence of non-NMDA receptor antagonism, stable locomotor activity (with lower cycle frequency) was also elicited by 5-HT, albeit with doses larger than in the control solution (15-40 microM). When NMDA and non-NMDA receptors were simultaneously blocked, 5-HT (5-120 microM) always failed to elicit locomotor activity. These data show that the operation of one glutamate receptor class was sufficient to express locomotor activity. As locomotor activity developed at a lower frequency than in the control solution after pharmacological block of either NMDA or non-NMDA receptors, it is suggested that both receptor classes were involved in locomotor pattern generation.     

Postnatal growth of corticospinal axons in the spinal cord of developing mice

The corticospinal tract (CST) plays an important role in the control of voluntary movements. Although the development of the CST has been studied extensively in other species, limited information is available on its development in mice. In the present study, the growth of corticospinal axons was characterized in developing mice using Phaseolus vulgaris leucoagglutinin (PHA-L). Our results indicate that the leading CST axons reach the 8th cervical segment at postnatal day (PD) 2, the 7th thoracic segment at PD4, the 13th thoracic segment at PD7, and the 5th lumbar segment at PD9. The arrival of corticospinal axons at the distal lumbar cord at PD9 was further confirmed by retrograde tracing using fast blue (FB). A waiting period of 2-3 days exists after the leading CST axons pass a particular segment before sending collaterals into the gray matter of that segment. The CST continues to increase in size in lower thoracic and lumbar areas up to PD14 when its adult appearance is achieved. In this study, the date of animal's sacrifice was used as the specific postnatal date to demonstrate the growth of the CST. This definition gives a more reliable indication of the exact location of the CST at a specific developmental time point since the CST continues to grow after tracer injections and since the dye is transported much faster than axonal growth. We suggest that these findings can be used as a template for studies on both normal and transgenic mice where some developmental significance is given to the CST.     

Localization of cyclooxygenase-2 and prostaglandin E2 receptor EP3 in the rat lumbar spinal cord

Cyclooxygenase-2 (COX-2) is now considered to be the major constitutively expressed COX isozyme in the central nervous system. The present immunocytochemical study details localization of COX-2 immunoreactivity in rat spinal cord along with the expression of prostaglandin E2 receptor subtype EP3. Prominent COX-2 staining was observed in the nuclear envelope of neurons throughout the spinal cord, especially in the superficial dorsal horn laminae and motoneurons of lamina IX, as well as in glial cells of the white matter. Expression of EP3 receptor was strictly confined to afferent terminal areas in the superficial dorsal horns.     

Human embryonic spinal cord grafts in adult rat spinal cord cavities: survival, growth, and interactions with the host

The ability of solid pieces of transplanted human embryonic spinal cord to survive, grow, and integrate with adult rat host spinal cord tissue was investigated. Unilateral cavities were surgically created at vertebral level T12-T13 in 10 athymic nude rats and 5 regular Sprague-Dawley rats. Seven of the athymic rats acutely received a human spinal cord graft, while the remaining 8 rats served as controls, with cavities alone. After 6 months the morphological outcome was evaluated with cresyl violet and with immunohistochemistry using antibodies toward human-specific neurofilament (hNF), human-specific Thy-1 (Thy-1), neurofilament, glial fibrillary acidic protein, serotonin (5-HT), and tyrosine hydroxylase (TH). The in situ morphology of the human embryonic spinal cord was also investigated and compared with grafts that were six months older. Solid human embryonic spinal cord grafts showed a 100% survival rate, grew to fill the volume of the cavity in a noninvasive manner, and expressed human specific antigens 6 months postgrafting. Thy-1 immunoreactivity (IR) was demonstrated up to 8 mm rostral to the graft suggestive of graft-derived fiber outgrowth. hNF-IR fibers and 5-HT- and TH-IR fibers traversed the graft-host border for a few hundred micrometers, respectively. Finally, our findings suggest that grafted solid pieces of human embryonic spinal cord minimize cystic deformations seen in the adult rat spinal cord with a unilateral cavity.     

Whole cell recordings of lumbar motoneurons during locomotor-like activity in the in vitro neonatal rat spinal cord

Whole cell current- and voltage-clamp recordings were obtained from lumbar motoneurons in the isolated neonatal rat spinal cord to characterize the behavior of motoneurons during neurochemically induced locomotor-like activity. Bath application of serotonin (10-100 muM) in combination with N-methyl-D-aspartate (1-12 muM) initially produced tonic membrane depolarization (mean = 26 mV), increased input resistance, decreased rheobase, and increased spike inactivation in response to depolarizing current pulse injections. After the initial tonic depolarization, rhythmic fluctuations of the motoneuron membrane potential (locomotor drive potentials; LDPs) developed that were modulated phasically in association with ventral root discharge. The peak and trough voltage levels of the LDP fluctuated above and below the membrane potential recorded immediately before the onset of rhythmic activity. Similarly, firing frequency was modulated above and below prelocomotion firing rates (in those motoneurons that displayed neurochemically induced tonic firing immediately before the onset of rhythmic activity). These observations are consistent with an alternation between phasic excitatory and inhibitory synaptic drives. The amplitude of LDPs and rhythmic excitatory drive current increased with membrane depolarization from -80 to -40 mV and then decreased with further depolarization, thus displaying nonlinear voltage-dependence. Faster frequency, small amplitude voltage fluctuations were observed superimposed on the depolarized phase of LDPs. In some motoneurons, the trajectory of these superimposed fluctuations was consistent with a synaptic origin, whereas in other cells, the regular sinusoidal appearance of the fluctuations and the occurrence of superimposed plateau potentials were more compatible with the activation of an intrinsic membrane property. One motoneuron displayed exclusively excitatory phasic drive, and another motoneuron was characterized by inhibitory phasic drive alone, during rhythmic activity. These findings are compatible with the concept of a central pattern generator that is capable of delivering both excitatory and inhibitory drive to motoneurons during locomotion. The data also suggest that the rhythmic excitatory and inhibitory outputs of the hypothetical half-center model can be dissociated and operate in isolation.     

Poly(alpha-hydroxyacids) for application in the spinal cord: resorbability and biocompatibility with adult rat Schwann cells and spinal cord

Future surgical strategies to restore neurological function in the damaged human spinal cord may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. We have studied the in vitro and in vivo degradability of various aliphatic polyesters as well as their effects on rat Schwann cells in vitro and on spinal cord tissue in vivo. In vitro, cylinders made of poly(D,L-lactic-co-glycolic acid) 50:50 (PLA25GA50) started to degrade at 7 days, compared with 28 days for cylinders made of poly(D,L-lactic acid) (PLA50). This faster degradation of PLA25GA50 was reflected by a much higher absorption of water. In vivo, after implantation of PLA25GA50 or PLA50 cylinders between the stumps of a completely transected adult rat spinal cord, the decrease in molecular weight of both polymers was similar to that found in vitro. In vitro degradation of poly(L-lactic acid) (PLA100) mixed with increasing amounts of PLA100 oligomers also was determined. The degradation rate of PLA100 mixed with 30% oligomers was found to be similar to that of PLA50. In vitro, PLA25GA50 and the breakdown products had no adverse effect on the morphology, survival, and proliferation of cultured rat Schwann cells. In vivo, PLA25GA50 cylinders were integrated into the spinal tissue 2 weeks after implantation, unlike PLA50 cylinders. At all time points after surgery, the glial and inflammatory response near the lesion site was largely similar in both experimental and control animals. At time points later than 1 week, neurofilament-positive fibers were found within PLA25GA50 cylinders or the remains thereof. Growth-associated protein 43, which is indicative of regenerating axons, was observed in fibers in the vicinity of the injury site and in the remains of PLA25GA50 cylinders. The results suggest that poly(alpha-hydroxyacids) are likely candidates for application in spinal cord regeneration paradigms involving Schwann cells.     

Non-conventional role of lysosomal acid phosphatase in olfactory receptor axons: co-localization with growth-associated phosphoprotein-43

Olfactory receptor neurons undergo a continuous turnover in adult mammals. It is largely unknown how their axons invade the olfactory bulb and induce synaptic re-organization in glomeruli. Here, the cytochemical localization of lysosomal acid phosphatase has been studied in olfactory bulbs of adult rats and mice. The enzyme has been identified by specific substrate, inhibitors and absence in lysosomal acid phosphatase-knockout mice. Lysosomal acid phosphatase is located in primary and secondary lysosomes, which are unevenly distributed in the olfactory nerve layer and among olfactory glomeruli. In consecutive sections of glomeruli, the intensity of lysosomal acid phosphatase immunoreactivity co-varied with that of growth-associated phosphoprotein. Electron microscopically, differential lysosomal acid phosphatase staining in glomeruli corresponded to different proportions of labelled and unlabelled axons. Quantification revealed that lysosomal acid phosphatase labelling was strongest in non-synaptic profiles of terminal axons, while it was weak in or even missing from most synaptic profiles. Hence, growing olfactory axons apparently carry more lysosomal acid phosphatase than those which have established synaptic contacts. Following olfactory deafferentation both lysosomal acid phosphatase activity and growth-associated phosphoprotein-43 are lost from glomeruli, suggesting that both proteins are expressed in olfactory sensory axons during growth, while lysosomal acid phosphatase is apparently not a marker of anterograde terminal degeneration.     

Characteristics of human fetal spinal cord grafts in the adult rat spinal cord: influences of lesion and grafting conditions

The present study evaluated the growth potential and differentiation of human fetal spinal cord (FSC) tissue in the injured adult rat spinal cord under different lesion and grafting conditions. Donor tissue at 6-9 weeks of gestational age was obtained through elective abortions and transplanted either immediately into acute resection (solid grafts) or into chronic contusion (suspension and solid grafts) lesions (i.e., 14-40 days after injury) in the thoracic spinal cord. The xenografts were then examined either histologically in plastic sections or immunocytochemically 1-3 months postgrafting. Intraspinal grafts in acute lesions demonstrated an 83% survival rate and developed as well-circumscribed nodules that were predominantly composed of immature astrocytes. Solid-piece grafts in chronic contusion lesions exhibited a 92% survival rate and also developed as nodular masses. These grafts, however, contained many immature neurons 2 months postgrafting. Suspension grafts in chronic contusion lesions had an 85% survival rate and expanded in a nonrestrictive, diffuse pattern. These transplants demonstrated large neuronally rich areas of neural parenchyma. Extensive neuritic outgrowth could also be seen extending from these grafts into the surrounding host spinal cord. These findings show that human FSC tissue reliably survives and differentiates in both acute and chronic lesions. However, both the lesion environment and the grafting techniques can greatly influence the pattern of differentiation and degree of host-graft integration achieved.     

Initial trajectories of sensory axons toward laminar targets in the developing mouse spinal cord

Quantitative ultrasonic tissue characterization using backscattered high-frequency intravascular ultrasound could provide a basis for the objective identification of lesions in vivo. Representation of local measurements of quantitative ultrasonic parameters in a conventional image format should facilitate their interpretation and thus increase their clinical utility. Toward this goal, the apparent integrated backscatter, the slope of attenuation (25-56 MHz) and the value of the attenuation on the linear fit at 37.5 MHz were measured using the backscattered radio frequency signals from in vitro human aortae. Local estimations of these ultrasonic parameters from both normal and atherosclerotic aortic segments were displayed in a B-scan format. The morphological features of these parametric images corresponded well to features of histological images of the same regions. The attenuation from 25-56 MHz of seven segments of the medial layer (both with and without overlying atheroma) were measured using the multinarrow-band backscatter method. The average attenuation in the media at 24 degrees C +/- 3 degrees C was 45 +/- 16 dB/cm at 25 MHz and 102 +/- 13 dB/cm at 50 MHz. This work represents progress toward the development of quantitative imaging methods for intravascular applications.     

Localization of last-order premotor interneurons in the lumbar spinal cord of rats

There is strong evidence that neural circuits underlying certain rhythmic motor behaviors are located in the spinal cord. Such local central pattern generators are thought to coordinate the activity of motoneurons through specific sets of last-order premotor interneurons that establish monosynaptic contacts with motoneurons. After injections of biotinylated dextran amine into the lateral and medial motor columns as well as the ventrolateral white matter at the level of the upper and lower segments of the lumbar spinal cord, we intended to identify and localize retrogradely labelled spinal interneurons that can likely be regarded as last-order premotor interneurons in rats. Regardless of the location of the injection site, labelled interneurons were revealed in laminae V-VIII along a three- or four-segment-long section of the spinal gray matter. Although most of the stained cells were confined to laminae V-VIII in all cases, the distribution of neurons within the confines of this area varied according to the site of injection. After injections into the lateral motor column at the level of the L4-L5 segments, the labelled neurons were located almost exclusively in laminae V-VII ipsilateral to the injection site, and the perikarya were distributed throughout the entire mediolateral extent of this area. Interneurons projecting to the lateral motor column at the level of the L1-L2 segments were also located in laminae V-VII, but most of them were concentrated in the middle one-third or in the lateral half of this area. Following injections into the medial motor column at the level of the L1-L2 segments, the majority of labelled neurons were confined to the medial aspect of laminae V-VII and lamina VIII, and the proportion of neurons that were found contralateral to the injection site was strikingly higher than in the other experimental groups. The results suggest that the organization of last-order premotor interneurons projecting to motoneurons, which are located at different areas of the lateral and medial motor columns and innervate different muscle groups, may present distinct features in the rat spinal cord.     

Three-dimensional analysis of the vascular system in the rat spinal cord with scanning electron microscopy of vascular corrosion casts. Part 1: Normal spinal cord

Vascular corrosion casts of polyester resin in the normal spinal cord at C4-C6 and C7-T1 were inspected three-dimensionally by scanning electron microscopy in 13 rats. Arteries and veins were easily differentiated by the impression pattern of endothelial nuclei on the casts. The centrifugal arterial system from the sulcal arteries supplied most of the gray and white matter in the ventral and lateral spinal cord. Each sulcal artery supplied only one side of the cord. The average number of sulcal arteries was 2.6 per mm. The centripetal arterial system from the posterior spinal arteries fed the posterior gray and white matter. In contrast with classical concepts, there was no pial arterial plexus on the ventral and ventrolateral surface except for infrequent transverse branches from the anterior spinal artery. In the posterior columns, two types of large veins were identified: the posterior medial septal veins and the posterior oblique veins that drained the posterior columns, medial posterior gray matter, and posterior gray commissure. The remainder of the gray and white matter was drained by the sulcal veins and the radial veins. This method clearly demonstrates the three-dimensional structure of both the arterial and venous system in the rat spinal cord.     

Immunohistochemical examination of intraspinal serotonin neurons and fibers in the chicken lumbar spinal cord and coexistence with Leu-enkephalin

This study compares Psychopathy Checklist-Revised (PCL-R) scores, DSM-III-R diagnoses, and select behavioral indices between hospitalized insanity acquittees (N = 18) and hospitalized insanity acquittees who successfully malingered (N = 18). The malingerers were significantly more likely to have a history of murder or rape, carry a diagnosis of antisocial personality disorder or sexual sadism, and produce greater PCL-R factor 1, factor 2, and total scores than insanity acquittees who did not malinger. The malingerers were also significantly more likely to be verbally or physically assaultive, require specialized treatment plans to control their aggression, have sexual relations with female staff, deal drugs, and be considered an escape risk within the forensic hospital. These findings are discussed within the context of insanity statutes and the relevance of malingering, psychopathy, and treatability to future policy concerning the disposition of insanity acquittees.