Characterization of A11 Neurons Projecting to the Spinal Cord of Mice

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons.     

Response of Cat Cortical Neurons to Position and Movement of the Femur

The contribution of joint afferents to the response of cortical neurons in area 3a to mechanical stimulation of the contralateral hindlimb was evaluated in cats anesthetized with sodium pentobarbital and paralyzed with pancuronium bromide. The hindlimb projection to the pericruciate cortex was established by recording the evoked potentials to electrical stimulation of the sciatic nerve and some of its branches, the biceps-semitendinosus and the quadratus femoris

Out of 169 neurons, 63 responded exclusively to cutaneous stimuli (superficial), whereas the others could be activated by local pressure of hindlimb muscles and/or by joint rotation (deep). Deep neurons were classified as slowly adapting (SA) or rapidly adapting (RA) units. In the neurons responding exclusively to joint rotation, the site of the receptive field could not be identified with certainty. In 13 deep neurons, their firing was affected by rotation of multiple joints of the contralateral hindlimb

In an attempt to identify the source of activation of cortical neurons, partial denervations and muscle disconnections were performed in five animals to isolate and stimulate the hip capsule. In these preparations, in 14 of 15 cortical neurons the source of activation was localized in the periarticular muscles, with no response to mechanical stimulation of the joint capsule. Only one neuron (S A) could be selectively excited by punctate pressure on the hip capsule

Our results suggest that in neurons of area 3a of the cat, the information about the position of the femur relies mainly on muscle afferents     

Cholinergic Receptor Alterations in the Brain Stem of Spinal Cord Injured Rats

Cholinergic receptors in upper motor neurons of brain stem control locomotion and coordination. Present study unravels cholinergic alterations in brain stem during spinal cord injury to understand signalling pathway changes which may be associated with spinal cord injury mediated motor deficits. We evaluated cholinergic function in brain stem by studying the expression of choline acetyl transferase and acetylcholine esterase. We quantified metabotropic muscarinic cholinergic receptors by receptor assays for total muscarinic, muscarinic M1 and M3 receptor subunits, gene expression studies using Real Time PCR and confocal imaging using FITC tagged secondary antibodies. The gene expression of ionotropic nicotinic cholinergic receptors and confocal imaging were also studied. The results from our study showed metabolic disturbance in cholinergic pathway as choline acetyl transferase is down regulated and acetylcholine esterase is up regulated in spinal cord injury group. The significant decrease in muscarinic receptors showed by decreased receptor number along with down regulated gene expression and confocal imaging accounts for dysfunction of metabotropic acetylcholine receptors in spinal cord injury group. Ionotropic acetylcholine receptor alterations were evident from the decreased gene expression of alpha 7 nicotinic acetylcholine receptors and confocal imaging. The motor coordination was analysed by Grid walk test which showed an increased foot slips in spinal cord injured rats. The significant reduction in brain stem cholinergic function might have intensified the motor dysfunction and locomotor disabilities.     

Effects of Dehydroepiandrosterone Sulfate on the Evoked Cortical Activity of Controls and of Brain-Injured Rats

1. Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are sex hormone precursors which exert marked neurotrophic and/or neuroprotective activity in the central nervous system (CNS).2. In the present electrophysiological experiments, we studied the effects of peripherally administered DHEAS on responses of the primary somatosensory (SSI) and motor cortices (MI) of (i) anesthetized controls and (ii) MI focal cold-lesioned rats. (iii) The effects of DHEAS on the field excitatory postsynaptic potentials (fEPSPs) were also studied in vitro brain slices. DHEAS (50 mg/kg) was injected subcutaneously 12 h before and immediately after cold lesion induction. The anesthetized rats were fixed in a stereotaxic frame, the SSI and MI were exposed, and control SSI and MI responses were evoked by contralateral whisker pad stimulation. After registration of the evoked responses for a 35-min period, a copper cylinder (2 mm in diameter) cooled with a mixture of acetone and dry ice (−78 °C) was applied to produce a lesion in the MI and the registration of the evoked responses was then continued for an additional 360 min.3. In the controls, DHEAS administration resulted in slight increases in amplitude of both the SSI and the MI responses. After focal cold lesion induction, the most significant reduction in amplitude was observed at the focus of the lesion in the primary MI, but the amplitudes of the SSI responses were also decreased. After 3–5 h of lesion induction, the amplitudes started to increase around the injury in the primary MI, while the SSI response had already started to recover 2 h after induction of the MI lesion. In the course of the postlesion recovery period, the MI responses peripherally to the center of the lesion frequently exhibited extremely high and low amplitudes. The paired-pulse paradigm revealed changing, but basically high levels of disinhibition and facilitation in extended cortical areas after focal cortical cold lesion induction. The deviations (e.g., the extremely augmented responses) in cortical functioning of the anesthetized rats were unambiguously diminished by DHEAS administration, and the period required for the cortical responses to recover was significantly shorter after the steroid treatment. In the in vitro studies, however, DHEAS administration resulted in an enhanced level of disinhibition in extended cortical areas of both the hemispheres.4. This observation draws attention to the possible differences between the results obtained in different models (in vitro vs. in situ). Nevertheless, all the presented data suggest that DHEAS treatment might have neuroprotective effect on the neocortex at least at a short-time scale.     

Brain and Spinal Cord Levels of Histamine in Lewis Rats with Acute Experimental Autoimmune Encephalomyelitis

Acute experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by inoculation with guinea pig spinal cord homogenate emulsified with Mycobacterium tuberculosis-enriched complete Freund's adjuvant (CFA). Control rats were inoculated with CFA alone. Control and EAE rats were killed on days 7, 9, 11, and 13 postinoculation, and regional brain and spinal cord levels of histamine were determined. No regional differences in histamine content between control and EAE rats were seen on day 7 or 9 postinoculation. However, depending on the region, EAE rats exhibited significantly higher levels of histamine in their CNS on day 11 or 13 postinoculation or on both. Thus, regionally and temporally specific increases in brain and spinal cord levels of histamine develop concomitant with or just after the appearance (on day 10 postinoculation) of clinical signs of acute EAE, a finding suggesting that histamine may be involved in the development or expression of acute EAE in Lewis rats.     

Stem Cells Downregulate the Elevated Levels of Tissue Plasminogen Activator in Rats After Spinal Cord Injury

We investigated the involvement of tPA after SCI in rats and effect of treatment with human umbilical cord blood derived stem cells. tPA expression and activity were determined in vivo after SCI in rats and in vitro in rat embryonic spinal neurons in response to injury with staurosporine, hydrogen peroxide and glutamate. The activity and/or expression of tPA increased after SCI and reached peak levels on day 21 post-SCI. Notably, the tPA mRNA activity was upregulated by 310-fold compared to controls on day 21 post-SCI. As expected, MBP expression is minimal at the time of peak tPA activity and vice versa. Implantation of hUCB after SCI resulted in the downregulation of elevated tPA activity/expression in vivo in rats as well as in vitro in spinal neurons. Our results demonstrated the involvement of tPA in the secondary pathogenesis after SCI as well as the therapeutic potential of hUCB.     

Set-Related Activity in the Premotor Cortex of Rhesus Monkeys: Effect of Triggering Cues and Relatively Long Delay Intervals

“Set-related activity” has been defined as a significant alteration in neuronal discharge rate during an “instructed delay period,” a period when a previously instructed movement is being withheld. It has been argued that set-related activity in the primate premotor cortex, or at least a significant proportion of it, reflects motor preparation. In most previous investigations, however, in which visual stimuli have triggered the movement and simultaneously indicated its target, set-related activity might reflect either the anticipation of or attention to the trigger stimulus. The present report shows that set-related activity is robust and can be directionally selective when trigger stimuli do not indicate the target and when a trigger stimulus is absent. Another feature of previous studies has been the relatively brief intervals between the instruction and trigger stimuli (typically 3 sec or less). In the present study, we were able to observe the activity of a small number of cells during longer delay periods. Set-related activity persists, although it becomes less consistent, for as much as 7.5 sec after an instruction stimulus. These results support the hypothesis that set-related activity reflects the preparation for specific limb movements.     

Repetitive Microstimulation Alters the Cortical Representation of Movements in Adult Rats

In order to examine the effects of repetitive stimulation on functional cortical organization, standard intracortical microstimulation (ICMS) techniques were used to generate maps of movement representations in motor cortex of rat. After identification of caudal and rostral forelimb fields and adjacent vibrissae and neck fields, one or more representational borders were defined in greater detail. Then a microelectrode was introduced into one of these representational fields, and ICMS current pulses were delivered at a rate of 1/sec for 1 to 3 hr. Following repetitive ICMS, significant changes in movement representations were observed using current levels that were either suprathreshold or subthreshold for evoking the site-specific movement. Electromyographic activity could be evoked at suprathreshold and near-threshold current levels, but not at the subthreshold current levels used here. Significant border shifts ranged from 210 to 670 μm. In each case in which shifts occurred, there appeared to be expansion of the movement represented at the repetitively stimulated site. The effects were progressive and reversible. These results suggest that at least under these unusual experimental circumstances, large representational changes can be generated very rapidly within motor cortex in the absence of any evident peripheral feedback.     

Chronological Observations of Primary Somatosensory Cortical Maps in Kittens following Low Thoracic (T12) Spinal Cord Transection at 2 Weeks of Age

The present study investigated the reorganization of the somatosensory cortex in kittens following T₁₂ spinal cord transection at 2 weeks of age. Multiunit electrophysiological methods were used to map the somatosensory cortex of kittens at 3, 6, and 9 weeks after the transection. The entire reorganized cortical region was driven by substitute cutaneous inputs, primarily from the trunk, at 3 weeks after spinal cord transection. Although the level of cortical responsiveness remained the same throughout the 9 weeks studied, internal trunk representation changed, and there was an increase in shoulder girdle representation and emergence of forelimb representation. Poor somatotopic and topographic order was observed in the reorganized cortex, regardless of time posttransection. Finally, trunk receptive fields displayed a wide variety of shapes, sizes, and orientations not seen in the normal cortex.     

Effects of Embryonic Neural Stem Cell Transplantation on DNA Damage in the Brain and Spinal Cord Following Spinal Cord Injury

Embryonic neural stem cell (ENSC) transplantation is used experimentally for the improvement of spinal cord repair following spinal cord injury (SCI). However, the effects of such intervention on oxidative stress and cell death remain unknown. We used in vivo Comet assay in the acute and chronic SCI groups compared with the SCI+ENSC transplantation groups of experimental rats in order to evaluate DNA damage in the spinal cord. Chronic SCI resulted in the generation of oxidative DNA damage in the spinal cord brain and kidneys, as indicated by high Comet assay parameters, including the percentage of DNA in the tail (T%, or TD), tail moment (TM), and tail length (TL). The DNA damage levels significantly decreased after ENSC transplantation in the spinal cords of acute and chronic SCI groups within the lesion site and rostrally and caudally to the injury, and in the brains and kidneys of the chronic SCI group. Thus, ENSC transplantation is found to be an effective tool for limitation of DNA damage following spinal cord injury.     

Neuronal Activity Preceding Directional and Nondirectional Cues in the Premotor Cortex of Rhesus Monkeys

Pre-cue activity, the neuronal modulation that precedes a predictable stimulus, was studied in the premotor cortex of three rhesus monkeys. In one condition, a directional cue dictated the timing and target of a forelimb movement. In another condition, a non-directional cue provided identical timing information but did not indicate the target. Of 501 task-related neurons recorded in premotor cortex, 168 showed pre-cue activity. The onset time of pre-cue activity varied markedly from trial to trial and cell to cell, ranging from trial initiation to 4.8 sec later. No pre-cue activity reflected the direction of limb movement; thus, the data argue against the hypothesis that pre-cue activity reflects preparation for specific limb movements. A small number of cells showed greater pre-cue activity before directional than before nondirectional cues, and this difference may reflect anticipation of the cue's directional information. However, the vast majority (84%) of neurons lacked such differences. We therefore hypothesize that most pre-cue activity reflects or contributes to a facet of behavior common to the two conditions: anticipation of the time and/or nature of events.     

Increased Uric Acid in the Developing Brain and Spinal Cord Following Cytomegalovirus Infection

Abstract: Tissue concentrations of uric acid were determined in the spinal cord, cerebellum, caudate-putamen, and cerebral cortex of developing mice following intraventricular inoculation with murine cytomegalovirus (MCMV) on postnatal day 10. Transient signs of neurological impairment were observed in MCMV-infected animals beginning on days 13–16 and continuing until days 19–21. At the onset of neurological impairment, uric acid concentrations in tissues from infected animals were 17–60-fold greater than in control animals. On postnatal day 70, 60 days after inoculation and 40 days after resolution of neurological signs, uric acid levels were still two- to threefold greater in infected animals. Histological examination revealed signs of focal ischemia in the cerebral and cerebellar cortices of MCMV-infected mice only at the onset of neurological impairment, with ischemic cell changes in some pyramidal neurons of the cerebral cortex. These results indicate that uric acid may be a sensitive marker of persistent vascular pathology resulting from cytomegalovirus infection of the developing nervous system     

Structural Types of Spinal Cord Marginal (Lamina I) Neurons Projecting to the Nucleus of the Tractus Solitarius in the Rat

The structural types of spinal cord marginal (lamina I) neurons projecting to the nucleus of the tractus solitarius (NTS) were studied. Upon injections of cholera toxin subunit B (CTb) into the caudal part of the NTS, including its lateral and medial portions, labeled cells occurred bilaterally in laminae I, IV-VII, and X, and the lateral spinal nucleus (LSN). After injections into the lateral portion alone, only a few cells were labeled in laminae V, VII, and X, and the LSN, and none in the superficial dorsal horn. Of 1882 labeled marginal cells, 38% belonged to the flattened type, 37% to the pyramidal type, and 25% to the fusiform type. Flattened and pyramidal cells were labeled in considerably greater numbers than those reported when other supraspinal targets of these cells were injected with CTb. Since cells in the NTS are known to be under marked 7-aminobutyric acidergic (GABA-ergic) inhibition, it is possible that only strong input conveyed by great numbers of flattened and pyramidal cells is capable of overcoming that barrier. Fusiform cells were labeled in numbers similar to those observed previously after tracer injections into the two other targets of this neuronal type, the parabrachial nuclei and the lateral reticular nucleus. Considering that these regions, as well as the NTS, control cardiovascular and respiratory functions, it is suggested that fusiform cells transmit noxious input that will influence autonomic reflexes processed in the three nuclei.     

Comparison of the Vascular Permeability of the Brain and the Spinal Cord to Mannitol and Inulin in Rats

The kinetics of the movement of [14C]mannitol and [14C]inulin from the blood into the CNS of the rat were measured. The spinal cord was found to have a higher permeability to these two substances than the brain. It is likely that the channels through which the tracers diffused are larger in the capillaries of the spinal cord than those in the brain.     

Localization and Central Projections of Primary Afferent Neurons That Innervate the Temporomandibular Joint in Cats

Primary afferent neurons that innervate the temporomandibular joint (TMJ) in cats were labeled by injecting a 2-5% solution of wheatgerm agglutinin bound to horseradish peroxidase into the joint capsule and capsular tissues in 14 cats and processing the brain stem and trigeminal ganglia using the tetramethylbenzidine method described by Mesulam (1978). The perikarya of ganglion cells that innervate the TMJ ranged in diameter from 15 to 109 μm and were primarily located in the posterolateral portion of the trigeminal ganglion. The central processes of these neurons entered the brain stem in middle pons and were distributed to all portions of the sensory trigeminal nuclei. However, the majority of labeled fibers and greatest density of terminal labeling were observed in the dorsal part of the main sensory nucleus and the subnucleus oralis of the spinal trigeminal nucleus. Very few labeled fibers were observed in the spinal tract of the trigeminal nerve below the obex. However, evidence for axon terminals was consistently observed in laminae I, II, and III of the medullary dorsal horn. These findings concur with physiological evidence showing that information from the TMJ influences neurons in rostral (Kawamura et al, 1967) and in caudal (Broton et al, 1985) portions of the trigeminal sensory nuclei.     

Thermostimulation-Induced Modulation of Conditioned Reflex Movement-Related Reactions of Cortical Neurons of the Cat

In awake cats trained to perform a food-procuring conditioned operant reflex (placing movement), we studied impulse reactions of 86 neurons of the motor cortex (field 4) related to realization of the above movements. As conditioning stimuli (CS) initiating the reflex, we used either non-noxious electrocutaneous stimulation (ECS) of the contralateral forelimb or an acoustic stimulus (sound click). Impulsation of cortical neurons was recorded under conditions of (i) isolated presentation of the CS (control), (ii) presentation of the CS (either ECS or acoustic stimulus) combined with thermostimulation (heating with a miniature electric bulb) of the skin of the working forelimb, and (iii) the same, but with stimulation of the resting forelimb. When we recorded spike activity of neurons within the projection motor zone of the resting limb subjected to ESC, alternating thermostimulation of both forelimbs resulted in considerable intensification and an increase in the duration of neuronal responses, especially in cases where thermostimulation was applied to the working limb ipsilateral to the recording site (a two- to threefold increase). When spike reactions were recorded within the motor cortex of the working forelimb, thermostimulation resulted in a considerable increase in the intensity of these reactions and a decrease in their latency, but only when such stimulation was applied to the working forelimb. Thermostimulation of the resting (ipsilateral, subjected to ESC) limb evoked opposite effects (the intensity of neuronal reactions dropped). In both situations, placing movements remained within the control limits. When sound click was used as a distant CS, thermostimulation of the working limb enhanced neuronal responses, increased their duration by 50-100%, and also increased the time of forestalling of the movement initiation by spike neuronal reactions. Thermostimulation of the resting forelimb in this situation also suppressed neuronal reactions. We conclude that foreign stimulations directed toward modifications of the receptor model of the operant reflex experimental situation formed in the animal result in a decrease in the intensity of the spike responses of field-4 neurons and prolongation of the latencies of these responses, while stimulations promoting the inflow of afferent information to the cortical projection of the working limb evoke opposite effects, an increase in the intensity of neuronal spike responses and a decrease in their latencies.     

Evidence for Synchronous Activation of Neurons Located in Different Layers of Primary Somatosensory Cortex

Although many studies have examined the columnar organization of primary somatosensory (SI) cortex, the functional relationship among neurons in different layers remains unclear. To understand how activity is coordinated among different cortical layers, the present investigation tested the hypothesis that the initial part of a peripheral stimulus produces a serial pattern of laminar activation in SI cortex. Extracellular discharges of 334 histologically recovered neurons were recorded from the medial bank of the coronal sulcus in nine anesthetized cats during electrical or cutaneous stimulation of the distal forelimb. Mean responses during the initial 50-msec period following stimulus onset were largest in layers IIIb or IV for both types of stimulation, but laminar differences in the magnitude of onset responses were not statistically significant. Among 175 neurons with responses exceeding 0.5 spikes per stimulus, electrical Stimulation consistently produced shorter response latencies than mechanical indentation in the extragranular (II, IIIa, V, VI), but not in the middle (IIIb, IV), cortical layers. The average minimum latencies for different cortical layers ranged from 7.4 to 10.1 msec for responses to electrical stimulation and from 10.3 to 11.6 msec for responses to mechanical indentations, but these laminar differences were not statistically significant. In some experiments, neurons in different layers of a cortical column were recorded simultaneously with dual-electrode assemblies; among 37 neuron pairs in which both neurons responded with more than 0.5 spikes per stimulus, response latencies were similar, even though the neurons were separated by several hundred microns. Cross-correlation analysis of the onset responses for neurons recorded simultaneously from different layers also indicated that many cells throughout a cortical column were activated nearly simultaneously by the initial phase of a peripheral stimulus. Results from the present study are compared with previous reports examining laminar patterns of activation.     

Dynamic Processing of Nociception in Cortical Network in Conscious Rats: A Laser-evoked Field Potential Study

(1) Field potential study in conscious rats provides a convenient and effective animal model for pain mechanism and pharmacological research. However, the spatial-temporal character of nociception processing in cortex revealed by field potential technique in conscious rats remains unclear. (2) In the present study, multi-channel field potentials evoked by noxious laser stimulation applied to the hind paw of conscious rats were recorded through 12 chronically implanted skull electrodes. Independent component analysis (ICA) was used to remove possible artifacts and to extract the specific nociception-related component. (3) Two fast sharp responses and one slow blunt response were evoked by noxious laser stimulation. Systemic morphine (5 mg/kg, i.p.) preferentially attenuated the amplitude of the slow blunt response while had no significant effect on the first two sharp responses. ICA revealed that those responses came from activities of contralateral anterior parietal area, medial frontal area and posterior parietal area. A movement artifact was also detected in this study. Partial directed coherence (PDC) analysis showed that there were changes of information flows from medial frontal and posterior parietal area to anterior parietal area after noxious laser stimulation. (4) Characterization of the spatio-temporal responses to noxious laser stimulation may be a valuable model for the study of pain mechanisms and for the assessment of analgesia.     

Calcium Enhances In Vitro Free Radical-Induced Damage to Brain Synaptosomes, Mitochondria, and Cultured Spinal Cord Neurons

Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+-free Krebs buffer resulted in a 27% inhibition of synaptosomal gamma-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 microM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+-ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+.