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c-Fos is used to identify system-wide neural activation with cellular resolution in vivo. However, c-Fos can only capture neural activation of one event. Targeted recombination in active populations (TRAP) allows the capture of two different c-Fos activation patterns in the same animal. So far, TRAP has only been used to examine brain circuits. This study uses TRAP to investigate spinal circuit activation during resting and stepping, giving novel insights of network activation during these events. The level of colabeled (c-Fos+ and TRAP+) neurons observed after performing two bouts of stepping suggests that there is a probabilistic-like phenomenon that can recruit many combinations of neural populations (synapses) when repetitively generating many step cycles. Between two 30-min bouts of stepping, each consisting of thousands of steps, only ∼20% of the neurons activated from the first bout of stepping were also activated by the second bout. We also show colabeling of interneurons that have been active during stepping and resting. The use of the FosTRAP methodology in the spinal cord provides a new tool to compare the engagement of different populations of spinal interneurons in vivo under different motor tasks or under different conditions.NEW & NOTEWORTHY The results are consistent with there being an extensive amount of redundancy among spinal locomotor circuits. Using the newly developed FosTRAP mouse model, only ∼20% of neurons that were active (labeled by Fos-linked tdTomato expression) during a first bout of 30-min stepping were also labeled for c-Fos during a second bout of stepping. This finding suggests variability of neural networks that enables selection of many combinations of neurons (synapses) when generating each step cycle.

The neural networks that generate stepping in complete spinal adult rats remain poorly defined. To address this problem we used c-fos (an activity-dependent marker) to identify active interneurons and motoneurons in the lumbar spinal cord of adult spinal rats during a 30-minute bout of bipedal stepping. Spinal rats were either step trained (30 min/day, 3 days/week for 7.5 weeks) or not step-trained. Stepping was enabled by epidural stimulation and the administration of the serotonergic agonists quipazine and 8-OHDPAT. A third group of spinal rats served as untreated (no stimulation, drugs, or stepping) controls. The number of activated cholinergic central canal cluster cells and partition neurons was higher in both step-trained and non-trained than untreated rats, and higher in non-trained than step-trained rats. The latter finding suggests that daily treatment with epidural stimulation plus serotonergic agonist treatment without step training enhanced the excitability of a broader cholinergic interneuronal population than step training. The number of activated interneurons in laminae II-VI of lumbar cross sections was higher in both step-trained and non-trained than untreated rats, and highest in step-trained rats. This finding suggests that this population of interneurons was responsive to epidural stimulation plus serotonergic treatment and that load-bearing induced when stepping had an additive effect. The number of activated motoneurons of all size categories was higher in the step-trained than the other two groups, reflecting a strong effect of loading on motoneuron recruitment. In general, these results indicate that the spinal networks for locomotion are similar with and without brain input.

We present a new perspective on the concept of feed-forward compared to feedback mechanisms for motor control. We propose that conceptually all sensory information in real time provided to the brain and spinal cord can be viewed as a feed-forward phenomenon. We also propose that the spinal cord continually adapts to a broad array of ongoing sensory information that is used to adjust the probability of making timely and predictable decisions of selected networks that will execute a given response. One interpretation of the term feedback historically entails responses with short delays. We propose that feed-forward mechanisms, however, range in timeframes of milliseconds to an evolutionary perspective, that is, “evolutionary learning.” Continuously adapting events enable a high level of automaticity within the sensorimotor networks that mediate “planned” motor tasks. We emphasize that either a very small or a very large proportion of motor responses can be under some level of conscious vs automatic control. Furthermore, we make a case that a major component of automaticity of the neural control of movement in vertebrates is located within spinal cord networks. Even without brain input, the spinal cord routinely uses feed-forward processing of sensory information, particularly proprioceptive and cutaneous, to continuously make fundamental decisions that define motor responses. In effect, these spinal networks may be largely responsible for executing coordinated sensorimotor tasks, even those under normal “conscious” control.

By using c-fos as an activity-dependent marker, we identified the cholinergic interneurons around the central canal and lumbar interneurons throughout the gray matter that were activated after a 30-min bout of quadrupedal treadmill stepping at a 0° or 25° incline in adult rats. Increased loading (elevated treadmill incline) imposed during treadmill stepping activated more cholinergic interneurons in the proximity of the central canal, i.e., central canal cluster cells and partition neurons. Since cholinergic central canal cells are thought to modulate motoneuron excitability, these data suggest that increased load during stepping may increase motoneuronal activity through activating more cholinergic central canal cells. We identified the muscle-specific motoneurons and afferent terminals in the spinal cord by injecting cholera toxin subunit B in the soleus and tibialis anterior muscles. The number of interneurons in lumbar segments L4 (tibialis anterior) and L5 (soleus) was higher in both groups that stepped on the treadmill compared with control and was highest in rats that stepped at a 25° incline. In a majority of laminae, the distribution of total and muscle-specific activated interneurons was highest in the 25° incline group and lowest in the control group for both muscles. These data could reflect increased peripheral (proprioceptive) input as well as supraspinal drive associated with stepping and demonstrate the differences in 1) the activation of cholinergic interneurons near the central canal and 2) the laminar and segmental location of interneurons throughout the gray matter that play a role in generating stepping under different loading conditions in adult rats.

Brain-derived neurotrophic factor (BDNF) induces plasticity within the lumbar spinal circuits thereby improving locomotor recovery in spinal cord-injured animals. We examined whether lumbar spinal cord motor neurons and other ventral horn cells of spinally transected (ST) rats were stimulated to produce BDNF mRNA in response to treadmill training. Rats received complete spinal cord transections as neonates (n=20) and one month later, received four weeks of either a low (100 steps/training session; n=10) or high (1000 steps/training session; n=10) amount of robotic-assisted treadmill training. Using combined non-radioactive in situ hybridization and immunohistochemical techniques, we found BDNF mRNA expression in heat shock protein 27-labeled motor neurons and in non-motor neuron cells was greater after 1000 steps/training session compared to the 100 steps/training session and was similar to BDNF mRNA labeling in untrained Intact rats. In addition, there were significantly more motor neurons that contained BDNF mRNA labeling within processes in the ST rats that received the higher amount of treadmill training. These findings suggested that motor neurons and other ventral horn cells in ST rats synthesized BDNF in response to treadmill training. The findings support a mechanism by which postsynaptic release of BDNF from motor neurons contributed to synaptic plasticity.

Rats receiving a complete spinal cord transection (ST) at a neonatal stage spontaneously can recover significant stepping ability, whereas minimal recovery is attained in rats transected as adults. In addition, neonatally spinal cord transected rats trained to step more readily improve their locomotor ability. We hypothesized that recovery of stepping in rats receiving a complete spinal cord transection at postnatal day 5 (P5) is attributable to changes in the lumbosacral neural circuitry and not to regeneration of axons across the lesion. As expected, stepping performance measured by several kinematics parameters was significantly better in ST (at P5) trained (treadmill stepping for 8 weeks) than age-matched non-trained spinal rats. Anterograde tracing with biotinylated dextran amine showed an absence of labeling of corticospinal or rubrospinal tract axons below the transection. Retrograde tracing with Fast Blue from the spinal cord below the transection showed no labeled neurons in the somatosensory motor cortex of the hindlimb area, red nucleus, spinal vestibular nucleus, and medullary reticular nucleus. Retrograde labeling transsynaptically via injection of pseudorabies virus (Bartha) into the soleus and tibialis anterior muscles showed no labeling in the same brain nuclei. Furthermore, re-transection of the spinal cord at or rostral to the original transection did not affect stepping ability. Combined, these results clearly indicate that there was no regeneration across the lesion after a complete spinal cord transection in neonatal rats and suggest that this is an important model to understand the higher level of locomotor recovery in rats attributable to lumbosacral mechanisms after receiving a complete ST at a neonatal compared to an adult stage.

The spinal cord is endogenously capable of several forms of adaptive plasticity and learning, including functional re-training, instrumental, and Pavlovian learning. Understanding the mechanisms of spinal plasticity could lead to improved therapies for spinal cord injury and other neuromotor disorders. We describe and demonstrate techniques for eliciting spinal learning in the adult mouse using the Horridge paradigm. In the Horridge paradigm, instrumental learning occurs when a nociceptive leg stimulus is made to be contingent on leg position and the spinal cord learns to maintain the ankle in a flexed position. Using fine-wire intramuscular stimulating electrodes, an inexpensive real-time video tracking system, and DC current stimulation, we were able to elicit instrumental spinal learning from mouse lumbrosacral spinal cords that were functionally isolated from the brain. This technique makes it more feasible to use the powerful genetic manipulations available in mice to better understand the processes of spinal learning, memory, and plasticity.

The serotonergic system is highly plastic, capable of adapting to changing afferent information in diverse mammalian systems. We hypothesized that removing supraspinal and/or peripheral input would play an important role in defining the distribution of one of the most prevalent serotonergic receptors, the 5-HT(1A) receptor (R), in the spinal cord. We investigated the distribution of this receptor in response to a complete thoracic (T7-T8) spinal cord transection (eliminating supraspinal input), or to spinal cord isolation (eliminating both supraspinal and peripheral input) in adult rats. Using two antibodies raised against either the second extracellular region (ECL(2)) or the third intracellular region (ICL(3)) of the 5-HT(1A)R, we compared the 5-HT(1A)R levels and distributions in specific laminae of the L3-L5 segments among the control, spinal cord-transected, and spinal cord-isolated groups. Each antibody labeled different populations of 5-HT(1A)R: ECL(2) labeled receptors in the axon hillock, whereas ICL(3) labeled receptors predominantly throughout the soma and proximal dendrites. Spinal cord transection increased the number of ECL(2)-positive cells in the medial region of laminae III-IV and lamina VII, and the mean length of the labeled axon hillocks in lamina IX. The number of ICL(3)-labeled cells was higher in lamina VII and in both the medial and lateral regions of lamina IX in the spinal cord-transected compared to the control group. In contrast, the length and number of ECL(2)-immunolabeled processes and ICL(3)-immunolabeled cells were similar in the spinal cord-isolated and control groups. Combined, these data demonstrate that the upregulation in 5-HT(1A)R that occurs with spinal cord transection alone is dependent on the presence of sensory input.

Trauma to the conus medullaris and cauda equina may result in autonomic, sensory, and motor dysfunctions. We have previously developed a rat model of cauda equina injury, where a lumbosacral ventral root avulsion resulted in a progressive and parallel death of motoneurons and preganglionic parasympathetic neurons, which are important for i.e. bladder control. Here, we report that an acute implantation of an avulsed ventral root into the rat conus medullaris protects preganglionic parasympathetic neurons and motoneurons from cell death as well as promotes axonal regeneration into the implanted root at 6 weeks post-implantation. Implantation resulted in survival of 44+/-4% of preganglionic parasympathetic neurons and 44+/-4% of motoneurons compared with 22% of preganglionic parasympathetic neurons and 16% of motoneurons after avulsion alone. Retrograde labeling from the implanted root at 6 weeks showed that 53+/-13% of surviving preganglionic parasympathetic neurons and 64+/-14% of surviving motoneurons reinnervated the graft. Implantation prevented injury-induced atrophy of preganglionic parasympathetic neurons and reduced atrophy of motoneurons. Light and electron microscopic studies of the implanted ventral roots demonstrated a large number of both myelinated axons (79+/-13% of the number of myelinated axons in corresponding control ventral roots) and unmyelinated axons. Although the diameter of myelinated axons in the implanted roots was significantly smaller than that of control roots, the degree of myelination was appropriate for the axonal size, suggesting normal conduction properties. Our results show that preganglionic parasympathetic neurons have the same ability as motoneurons to survive and reinnervate implanted roots, a prerequisite for successful therapeutic strategies for autonomic control in selected patients with acute conus medullaris and cauda equina injuries.

Adult monkey primary visual cortex contains a diverse population of stellate neurons that utilize the neurotransmitter gamma aminobutyric acid (GABA). Two glutamic acid decarboxylase (GAD) enzymes that synthesize GABA, GAD65 and GAD67, were localized within these stellate neurons by in situ hybridization of 35S or digoxigenin (DIG) labeled riboprobes. Double labels were done by using 35S GAD67 riboprobe and GABA immunocytochemistry on the same section to verify that the neuronal population identified by immunocytochemistry was the same one studied in the in situ hybridization experiments. We find that GAD65 mRNA and GAD67 mRNA are widely distributed in the cortex, with four bands of heavily labeled neurons in upper layer 2, lower 3, 4C, and 6. GAD67 labeled neurons were more obvious in layer 4C beta, while GAD65 containing neurons were common in layer 1 and white matter. Northern blots and in situ hybridization on sections with both 35S and DIG riboprobes indicate that cortical neurons typically contain more GAD67 mRNA. Cell counts show that 18% of all cortical neurons contain GAD67 mRNA and 13% contain GAD65 mRNA, suggesting that a small population of GABA neurons might lack GAD65. Cell bodies that contain high amounts of GAD65 mRNA are prominent in layers deep 3, 4B, 4C alpha, and 6 and often are the largest cells in their respective layers. Double labels demonstrate that 96% of all GABA+ neurons contain GAD67 mRNA. Neurons heavily labeled for GABA tend to have smaller cell bodies and contain less GAD67 mRNA, while lightly labeled GABA neurons are larger and contain more GAD67 mRNA. These data indicate that most GABA neurons in monkey striate cortex contain both GAD enzymes. Although the differences in GABA content, cell size, laminar distribution, and GAD mRNA concentration suggest different requirements for GAD67 and GAD65 in cortical circuits, our experiments do not reveal what different roles these two enzymes subserve within GABAergic stellate neurons.

The number of cerebellar Purkinje cells is increased by over 40% in young transgenic mice that overexpress a human Bcl-2 transgene (Hu-Bcl-2). To determine whether the Bcl-2-mediated rescue of Purkinje cells persists through life, the numbers of Purkinje cells were estimated in 6-, 12-, 18-, and 24-month-old Hu-Bcl-2 transgenic mice and age-matched controls. In addition, the expression of four markers for Purkinje cell differentiation, calbindin (CaBP), the 67-kDa isoform of glutamic acid decarboxylase (GAD67), gamma-aminobutyric acid transaminase (GABA-T), and the NMDA-R1 receptor subtype (NMDA-NR1) was analyzed in 6-month-old Hu-Bcl-2 transgenics and controls to determine whether overexpression of Bcl-2 and rescue from naturally occurring cell death affects the normal differentiation of Purkinje cells. The estimates of Purkinje cell numbers showed that the number of Purkinje cells in the Hu-Bcl-2 transgenics declines after 6 months to approach wild-type values by 18 months. Although the exogenous human BCL-2 is still expressed in Purkinje cells at 24 months, the expression levels of human BCL-2 appear to decline significantly after 6 months, suggesting that survival of the supernumary Purkinje cells depends on the sustained overexpression of Bcl-2. All the Purkinje cells in the Hu-Bcl-2 transgenic mice appeared to express normal levels of the differentiation markers analyzed so there was no evidence for a class of Purkinje cells that do not differentiate normally when rescued from naturally occurring cell death.

The inhibitory neurotransmitter GABA is synthesized by glutamic acid decarboxylase (GAD), and two isoforms of this enzyme exist: GAD65 and GAD67. Immunocytochemical studies of the spinal cord have shown that whilst both are present in the dorsal horn, GAD67 is the predominant form in the ventral horn. The present study was carried out to determine the pattern of coexistence of the two GAD isoforms in axonal boutons in different laminae of the cord, and also to examine the relation of the GADs to the glycine transporter GLYT2 (a marker for glycinergic axons), since many spinal neurons are thought to use GABA and glycine as co-transmitters. Virtually all GAD-immunoreactive boutons throughout the spinal grey matter were labelled by both GAD65 and GAD67 antibodies; however, the relative intensity of staining with the two antibodies varied considerably. In the ventral horn, most immunoreactive boutons showed much stronger labelling with the GAD67 antibody, and many of these were also GLYT2 immunoreactive. However, clusters of boutons with high levels of GAD65 immunoreactivity were observed in the motor nuclei, and these were not labelled with the GLYT2 antibody. In the dorsal horn, some GAD-immunoreactive boutons had relatively high levels of labelling with either GAD65 or GAD67 antibody, whilst others showed a similar degree of labelling with both antibodies. GLYT2 immunoreactivity was associated with many GAD-immunoreactive boutons; however, this did not appear to be related to the pattern of GAD expression. It has recently been reported that there is selective depletion of GAD65, accompanied by a loss of GABAergic inhibition, in the ipsilateral dorsal horn in rats that have undergone peripheral nerve injuries [J Neurosci 22 (2002) 6724]. Our finding that some boutons in the superficial laminae showed relatively high levels of GAD65 and low levels of GAD67 immunoreactivity is therefore significant, since a reduction in GABA synthesis in these axons may contribute to neuropathic pain.

C6 cells express proteins and mRNAs that are characteristic of both glia and neurons. Agents that increase intracellular levels of cyclic AMP (cAMP) decrease the enzymatic activity of glutamate decarboxylase (GAD), a neuronal marker, and the mRNA levels for one of the two GAD isoenzymes, GAD67. This reduction is accompanied by increased levels of glial fibrillary acidic protein (GFAP) mRNA, an astrocyte marker. Transient transfection assays, in which a 2-kb upstream regulatory region of the human GFAP gene was linked to a reporter gene, indicate that at least some of the cAMP-mediated increase of GFAP mRNA levels is due to increased transcription. Increases in intracellular cAMP appear to induce differentiation of C6 cells toward a more mature astrocyte phenotype.

Chronic administration of ethanol to rats on an intermittent regimen, for 60 repeated intoxicating doses and repeated withdrawal episodes, results in a long-lasting kindling phenomenon. This involves an increasing severity of withdrawal, including a reduced threshold to seizures produced by the GABA(A) antagonist, pentylenetetrazol. We have shown previously that muscimol-evoked 36Cl- efflux and paired-pulse inhibition (involving GABA(A)-mediated recurrent inhibition) were decreased persistently in the CA1 region of hippocampal slices from chronic intermittent ethanol (CIE)-treated rats. We now report elevated levels of mRNA in forebrain for the alpha4 subunit of the GABA(A) receptor (GABAR), considered to be a constituent of pharmacologically and physiologically novel subtypes of GABARs. Using in situ hybridization with digoxigenin-labeled RNA probes, we show that at 2 days withdrawal, 60-dose CIE leads to a significant 30% increase in alpha4 subunit mRNA levels in the dentate gyrus, 46% increase in the CA3, and 26% increase in the CA1 regions. In contrast, there was no significant change in the mRNAs for the alpha5 subunit or glutamic acid decarboxylase 67 in the same regions. This study suggests that GABAR subunit-selective alterations occur after CIE treatment, possibly resulting in the alteration of the subunit composition of GABARs, with presumably altered physiological functions. This plasticity of GABARs may contribute to the increased withdrawal severity, reduced hippocampal inhibition, and increased seizure susceptibility of this animal model of human alcohol dependence.

The present review presents a series of concepts that may be useful in developing rehabilitative strategies to enhance recovery of posture and locomotion following spinal cord injury. First, the loss of supraspinal input results in a marked change in the functional efficacy of the remaining synapses and neurons of intraspinal and peripheral afferent (dorsal root ganglion) origin. Second, following a complete transection the lumbrosacral spinal cord can recover greater levels of motor performance if it has been exposed to the afferent and intraspinal activation patterns that are associated with standing and stepping. Third, the spinal cord can more readily reacquire the ability to stand and step following spinal cord transection with repetitive exposure to standing and stepping. Fourth, robotic assistive devices can be used to guide the kinematics of the limbs and thus expose the spinal cord to the new normal activity patterns associated with a particular motor task following spinal cord injury. In addition, such robotic assistive devices can provide immediate quantification of the limb kinematics. Fifth, the behavioural and physiological effects of spinal cord transection are reflected in adaptations in most, if not all, neurotransmitter systems in the lumbosacral spinal cord. Evidence is presented that both the GABAergic and glycinergic inhibitory systems are up-regulated following complete spinal cord transection and that step training results in some aspects of these transmitter systems being down-regulated towards control levels. These concepts and observations demonstrate that (a) the spinal cord can interpret complex afferent information and generate the appropriate motor task; and (b) motor ability can be defined to a large degree by training.

To determine changes in γ-aminobutyric acid (GABA) in the spinal cord in response to a complete transection, we examined the cellular and tissue changes of the two forms of GABA synthetic enzyme glutamate decarboxylase (GAD65 and GAD67). In situ hybridization, immunohistochemistry, and Western blot analyses show that spinal cord transection between thoracic segments 12 and 13 results in an increase of GAD67, but not GAD65, protein and mRNA in the lumbar spinal cord. This increase occurs mainly in the dorsal horn and persists for at least 12 months. In addition, there was relatively high GAD67-immunoreactivity around the central canal, with dorsolateral GAD67-immunoreactive fibers extending toward the ependyma and into the central canal in the transected animals. We suggest that an increase in GAD67 leads to increased GABA production in spinal neurons below the injury site, resulting in altered inhibition and trophic support during posttrauma recovery and adaptation. Increased GABA synthesis around the central canal, in the vicinity of ependymal cells, may represent part of a regenerative process in the mammalian spinal cord, reminiscent of that observed in lower vertebrates. J. Neurosci. Res. 60:219–230, 2000 © 2000 Wiley-Liss, Inc.

Modification of GABAA receptor mRNA levels by seizure activity can regulate general neuronal excitability. The possibility of absence seizure-induced alteration in GABAA receptor alpha 1, alpha 4, beta 2, and gamma 2 subunit gene expression in thalamic relay nuclei was studied in a rat model of absence seizures induced by gamma-hydroxybutyric acid (GHB). We observed a marked increase in alpha 1 mRNA and a corresponding decrease in alpha 4 mRNA in thalamic relay nuclei 2-4 h after the onset of GHB-induced absence seizures (when the seizures were terminating). These changes were selective to these alpha isoforms as neither beta 2 nor gamma 2 mRNA changed following seizures and occurred only in thalamic relay nuclei but not in hippocampus, a structure from which absence seizures do not evolve. The alterations in alpha 1 and alpha 4 mRNA persisted until about 12 h, and by 24 h after the seizure-onset the mRNA levels normalized. Blocking GHB-seizures produced no change in the levels of alpha 1 and alpha 4 mRNA in thalamic relay nuclei, suggesting that seizures themselves were responsible for mRNA alterations. In order to determine if absence seizure-induced changes in alpha 1 and alpha 4 mRNA had any physiological significance, GHB was readministered in rats 6 and 24 h after the onset of seizures. The total duration of GHB-seizures was found to be significantly decreased when GHB was readministered at 6 h but not 24 h after the seizure-onset. These results suggest that absence seizures regulate GABAA receptor alpha 1 and alpha 4 gene expression in thalamic relay nuclei as a compensatory mechanism by which absence seizures are terminated.

Can lower limb motor function be improved after a spinal cord lesion by re-engaging functional activity of the upper limbs? We addressed this issue by training the forelimbs in conjunction with the hindlimbs after a thoracic spinal cord hemisection in adult rats. The spinal circuitries were more excitable, and behavioural and electrophysiological analyses showed improved hindlimb function when the forelimbs were engaged simultaneously with the hindlimbs during treadmill step-training as opposed to training only the hindlimbs. Neuronal retrograde labelling demonstrated a greater number of propriospinal labelled neurons above and below the thoracic lesion site in quadrupedally versus bipedally trained rats. The results provide strong evidence that actively engaging the forelimbs improves hindlimb function and that one likely mechanism underlying these effects is the reorganization and re-engagement of rostrocaudal spinal interneuronal networks. For the first time, we provide evidence that the spinal interneuronal networks linking the forelimbs and hindlimbs are amenable to a rehabilitation training paradigm. Identification of this phenomenon provides a strong rationale for proceeding toward preclinical studies for determining whether training paradigms involving upper arm training in concert with lower extremity training can enhance locomotor recovery after neurological damage.

Given that the spinal cord is capable of learning sensorimotor tasks and that dietary interventions can influence learning involving supraspinal centers, we asked whether the presence of omega-3 fatty acid docosahexaenoic acid (DHA) and the curry spice curcumin (Cur) by themselves or in combination with voluntary exercise could affect spinal cord learning in adult spinal mice. Using an instrumental learning paradigm to assess spinal learning we observed that mice fed a diet containing DHA/Cur performed better in the spinal learning paradigm than mice fed a diet deficient in DHA/Cur. The enhanced performance was accompanied by increases in the mRNA levels of molecular markers of learning, i.e., BDNF, CREB, CaMKII, and syntaxin 3. Concurrent exposure to exercise was complementary to the dietary treatment effects on spinal learning. The diet containing DHA/Cur resulted in higher levels of DHA and lower levels of omega-6 fatty acid arachidonic acid (AA) in the spinal cord than the diet deficient in DHA/Cur. The level of spinal learning was inversely related to the ratio of AA:DHA. These results emphasize the capacity of select dietary factors and exercise to foster spinal cord learning. Given the non-invasiveness and safety of the modulation of diet and exercise, these interventions should be considered in light of their potential to enhance relearning of sensorimotor tasks during rehabilitative training paradigms after a spinal cord injury.

Two isoforms of glutamic acid decarboxylase (GAD67 and GAD65) and their mRNAs were localized in the rat brain by immunohistochemistry and nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes. In most brain regions, both GAD isoforms were present in neuronal cell bodies as well as axon terminals. A few populations of neurons, such as those in the reticular nucleus of the thalamus, exhibited similar cell body labeling for both GADs. However, in many brain regions, the cell bodies that were immunoreactive for GAD67 were often more numerous than those that were immunoreactive for GAD65. In contrast, the density (quantity) of GAD65-immunoreactive axon terminals was higher than that of GAD67-immunoreactive terminals. Strong parallels were observed between the intensity of immunohistochemical labeling of cell bodies and the levels of mRNA labeling for both GAD isoforms. Many groups of GAD-containing cell bodies were distinctly labeled for GAD67, and these same groups of neurons were heavily labeled for GAD67 mRNA. Such neurons included Purkinje cells of the cerebellar cortex, nonpyramidal cells in the cerebral cortex, and neurons of the reticular nucleus of the thalamus. Similar parallels in labeling were observed for GAD65 and its mRNA. Distinct cell body labeling for the protein and associated high levels of GAD65 mRNA were found in neurons of the reticular nucleus of the thalamus and periglomerular cells in the olfactory bulb. However, many cell bodies were not readily labeled for GAD65 with immunohistochemical methods. Such absence or weakness of cell body labeling for the protein was associated with low or moderate levels of GAD65 mRNA. Even though light cell body staining was frequently observed for GAD65 and its mRNA, strong axon terminal labeling for GAD65 was present. Thus, in the deep cerebellar nuclei to which the Purkinje cells of the cerebellar cortex project, strong terminal labeling was observed for both GAD isoforms even though only light cell body labeling of the Purkinje cells was obtained for GAD65 and its mRNA. The findings suggest that the two isoforms of GAD are present in most classes of GABA neurons but that they are not similarly distributed within the neurons. GAD67 is present in readily detectable amounts in many GAD-containing cell bodies whereas GAD65 is particularly prominent in many axon terminals. In addition, neurons that express either form of GAD mRNA also express the corresponding protein. Levels of labeling for the GAD mRNAs suggest that, under normal conditions, the synthesis of GAD65 is frequently lower than that of GAD67.(ABSTRACT TRUNCATED AT 400 WORDS)

Mice that are deficient in classical major histocompatibility complex class I (MHCI) have abnormalities in synaptic plasticity and neurodevelopment and have more extensive loss of synapses and reduced axon regeneration after sciatic nerve transection, suggesting that MHCI participates in maintaining synapses and axon regeneration. Little is known about the biological consequences of up-regulating MHCI's expression on neurons. To understand MHCI's neurobiological activity better, and in particular its role in neurorepair after injury, we have studied neurorepair in a transgenic mouse model in which classical MHCI expression is up-regulated only on neurons. Using a well-established spinal cord injury (SCI) model, we observed that transgenic mice with elevated neuronal MHCI expression had significantly better recovery of locomotor abilities after SCI than wild-type mice. Although previous studies have implicated inflammation as both deleterious and beneficial for recovery after SCI, our results point directly to enhanced neuronal MHCI expression as a beneficial factor for promoting recovery of locomotor function after SCI.

gamma-Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5'-phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission.

GABA signaling plays an important role in the spinal cord response to injury and subsequent motor training. Since benzodiazepines are commonly used to treat muscle spasticity in spinal cord injured subjects and the gamma2 subunit of the GABA(A) receptor is necessary for benzodiazepine binding, this subunit may be an important factor modulating sensorimotor function after an injury. Changes in gamma2 levels in muscle-specific motoneurons and surrounding astrocytes were determined approximately 3 months after a complete mid-thoracic spinal cord transection at P5 in non-trained and in step-trained spinal rats. Soleus (ankle extensor) and tibialis anterior (TA, ankle flexor) motor pools were identified using retrograde labeling via intramuscular injections of Fast Blue or Fluoro Gold, respectively. Lumbar spinal cord sections showed gamma2 immunostaining in both soleus and TA motoneurons and astrocytes. gamma2 immunoreactivity on the soma of soleus and TA motoneurons in spinal rats was differentially modulated. Compared to intact rats, spinal rats had higher levels of gamma2 in TA, and lower levels in soleus motoneurons. Step training restored GABA(A) gamma2 levels towards control values in motoneuronal pools of both muscles. In contrast, the gamma2 levels were elevated in surrounding astrocytes of both motor pools in spinal rats, and step training had no further effect. Thus, motor training had a specific effect on those neurons that were directly involved with the motor task. Since the gamma2 subunit is involved with GABA(A) receptor trafficking and synaptic clustering, it appears that this subunit could be an important component of the activity-dependent response of the spinal cord after a spinal injury.

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.

For a complete adult spinal rat to regain some weight-bearing stepping capability, it appears that a sequence of specific proprioceptive inputs that are similar, but not identical, from step to step must be generated over repetitive step cycles. Furthermore, these cycles must include the activation of specific neural circuits that are intrinsic to the lumbosacral spinal cord segments. For these sensorimotor pathways to be effective in generating stepping, the spinal circuitry must be modulated to an appropriate excitability level. This level of modulation is sustained from supraspinal input in intact, but not spinal, rats. In a series of experiments with complete spinal rats, we have shown that an appropriate level of excitability of the spinal circuitry can be achieved using widely different means. For example, this modulation level can be acquired pharmacologically, via epidural electrical stimulation over specific lumbosacral spinal cord segments, and/or by use-dependent mechanisms such as step or stand training. Evidence as to how each of these treatments can “tune” the spinal circuitry to a “physiological state” that enables it to respond appropriately to proprioceptive input will be presented. We have found that each of these interventions can enable the proprioceptive input to actually control extensive details that define the dynamics of stepping over a range of speeds, loads, and directions. A series of experiments will be described that illustrate sensory control of stepping and standing after a spinal cord injury and the necessity for the “physiological state” of the spinal circuitry to be modulated within a critical window of excitability for this control to be manifested. The present findings have important consequences not only for our understanding of how the motor pattern for stepping is formed, but also for the design of rehabilitation intervention to restore lumbosacral circuit function in humans following a spinal cord injury.

Adult male rats were lesioned with 6-hydroxydopamine in order to destroy the nigrostriatal dopaminergic projections. In rats with such a lesion, we found a parallel increase in glutamic acid decarboxylase (GAD) activity and GAD mRNA in the striatum ipsilateral to the lesion at 4 weeks and 4 months after the lesion. These observations support the proposal that nigral dopaminergic neurons exert a tonic inhibitory control over the striatal GABAergic neurons. Our observations also suggest that the dopaminergic neurons can inhibit gene expression in striatal GABAergic neurons and that the enhanced striatal GAD activity following lesions of the dopaminergic projections is due to 'de novo' synthesis of the enzyme.

The effect of two chronic motor training paradigms on the ability of the lumbar spinal cord to perform an acute instrumental learning task was examined in neonatally (postnatal day 5; P5) spinal cord transected (i.e., spinal) rats. At approximately P30, rats began either unipedal hindlimb stand training (Stand-Tr; 20-25min/day, 5days/week), or bipedal hindlimb step training (Step-Tr; 20min/day; 5days/week) for 7 weeks. Non-trained spinal rats (Non-Tr) served as controls. After 7 weeks all groups were tested on the flexor-biased instrumental learning paradigm. We hypothesized that (1) Step-Tr rats would exhibit an increased capacity to learn the flexor-biased task relative to Non-Tr subjects, as locomotion involves repetitive training of the tibialis anterior (TA), the ankle flexor whose activation is important for successful instrumental learning, and (2) Stand-Tr rats would exhibit a deficit in acute motor learning, as unipedal training activates the ipsilateral ankle extensors, but not flexors. Results showed no differences in acute learning potential between Non-Tr and Step-Tr rats, while the Stand-Tr group showed a reduced capacity to learn the acute task. Further investigation of the Stand-Tr group showed that, while both the ipsilateral and contralateral hindlimbs were significantly impaired in their acute learning potential, the contralateral, untrained hindlimbs exhibited significantly greater learning deficits. These results suggest that different types of chronic peripheral input may have a significant impact on the ability to learn a novel motor task, and demonstrate the potential for experience-dependent plasticity in the spinal cord in the absence of supraspinal connectivity.

An investigation of c-fos activation pattern in spinal neurons of intact adult rats after acute bouts of treadmill locomotion.To map spinal neurons that are involved in quadrupedal treadmill stepping of intact adult rats by using c-fos as a marker.Los Angeles, CA, USA.Spinal cord sections of rats that were not stepped (n = 4) were used to map the FOS-positive (+) neurons under basal conditions. The stepped group (n = 16) was placed on a treadmill to step quadrupedally for varying durations to induce c-fos activity. Spinal cord sections of thoracic and lumbar segments of Stp and Nstp rats were processed using a c-fos antibody, choline acetyl transferase and heat shock protein 27 for identifying motoneurons.Stepping induced a greater number of FOS+ neurons than was observed in rats that did not step on the treadmill. There was a rostrocaudal and a dorsoventral gradient of FOS labeled neurons. The number of FOS+ neurons increased with the duration of treadmill stepping. Significant increases in FOS+ neurons were in the most medial parts of laminae IV, V, and VII. FOS+ motoneurons increased with treadmill stepping, particularly in large motoneurons (or = 700 microm2).These data suggest that FOS can be used to identify activity-dependent neuronal pathways in the spinal cord that are associated with treadmill stepping, specifically in lamina VII and in alpha motoneurons.NIH NS16333, NS40917, and the Christopher Reeve Paralysis Foundation (CRPF VEC 2002).

▪ Abstract Motor function is severely disrupted following spinal cord injury (SCI). The spinal circuitry, however, exhibits a great degree of automaticity and plasticity after an injury. Automaticity implies that the spinal circuits have some capacity to perform complex motor tasks following the disruption of supraspinal input, and evidence for plasticity suggests that biochemical changes at the cellular level in the spinal cord can be induced in an activity-dependent manner that correlates with sensorimotor recovery. These characteristics should be strongly considered as advantageous in developing therapeutic strategies to assist in the recovery of locomotor function following SCI. Rehabilitative efforts combining locomotor training pharmacological means and/or spinal cord electrical stimulation paradigms will most likely result in more effective methods of recovery than using only one intervention.

In this chapter we examine the recovery of posture and locomotion in mammals fol1owing a complete spinal cord lesion. The mechanisms by which the ability to stand and step can be regained fol1owing a complete injury is described with respect to central pattern generation, processing of sensory (proprioceptive and cutaneous) input by the spinal cord circuitry and the concept of automaticity. A series of intervention strategies designed to enhance motor function after a complete spinal cord lesion are examined, i.e., motor training and spinal cord learning, electrical stimulation of nerves, muscles, and the spinal cord below the level of the lesion, pharmacological facilitation of motor function, administration of growth/neurotrophic factors, and several types of cell and tissue implants to induce axonal growth. The general conclusion is that a significant level of motor function can be recovered after a complete spinal cord lesion. Although motor training has been shown to be one of the most effective strategies to date, a number of other interventions also are beginning to produce significant improvements in motor function. Some of these improvements can be attributed solely to functional reorganization of the spinal cord circuitry distal to the lesion, whereas other interventions appear to have been mediated by renewed connectivity between the neural elements above and below the lesion site. There seems to be a general consensus that a greater level of motor recovery is likely to be attained by a combination of therapies.

Injuries to the cauda equina of the spinal cord result in autonomic and motor neuron dysfunction. We developed a rodent lumbosacral ventral root avulsion injury model of cauda equina injury to investigate the lesion effect in the spinal cord. We studied the retrograde effects of a unilateral L5-S2 ventral root avulsion on efferent preganglionic parasympathetic neurons (PPNs) and pelvic motoneurons in the L6 and S1 segments at 1, 2, 4, and 6 weeks postoperatively in the adult male rat. We used Fluoro-Gold-prelabeling techniques, immunohistochemistry, and quantitative stereologic analysis to show an injury-induced progressive and parallel death of PPNs and motoneurons. At 6 weeks after injury, only 22% of PPNs and 16% of motoneurons remained. Furthermore, of the neurons that survived at 6 weeks, the soma volume was reduced by 25% in PPNs and 50% in motoneurons. Choline acetyltransferase (ChAT) protein was expressed in only 30% of PPNs, but 80% of motoneurons remaining at 1 week postoperatively, suggesting early differential effects between these two neuronal types. However, all remaining PPNs and motoneurons were ChAT positive at 4 weeks postoperatively. Nuclear condensation and cleaved caspase-3 were detected in axotomized PPNs and motoneurons, suggesting apoptosis as a contributing mechanism of the neural death. We conclude that lumbosacral ventral root avulsions progressively deplete autonomic and motor neurons. The findings suggest that early neuroprotection will be an important consideration in future attempts of treating acute cauda equina injuries.

The ability to perform stepping and standing can be reacquired after complete thoracic spinal cord transection in adult cats with appropriate, repetitive training. We now compare GAD(67)A levels in the spinal cord of cats that were trained to step or stand. We confirmed that a complete spinal cord transection at approximately T12 increases glutamic acid decarboxylase (GAD)(67) in both the dorsal and ventral horns of L5-L7. We now show that step training decreases these levels toward control. Kinematic analyses show that this downward modulation is correlated inversely with stepping ability. Compared with intact cats, spinal cord-transected cats had increased punctate GAD(67) immunoreactivity around neurons in lamina IX at cord segments L5-L7. Compared with spinal nontrained cats, those trained to stand on both hindlimbs had more GAD(67) puncta bilaterally in a subset of lamina IX neurons. In cats trained to stand unilaterally, this elevated staining pattern was limited to the trained side and extended for at least 4 mm in the L6 and L7 segments. The location of this asymmetric GAD(67) staining corresponded to the motor columns of primary knee flexors, which are minimally active during standing, perhaps because of extensor-activated inhibitory interneuron projections. The responsiveness to only a few days of motor training, as well as the GABA-synthesizing potential in the spinal cord, persists for at least 25 months after the spinal cord injury. This modulation is specific to the motor task that is performed repetitively and is closely linked to the ability of the animal to perform a specific motor task.

4-Aminobutyric acid (GABA), a major inhibitory neurotransmitter of mammalian central nervous system, is found in a wide range of organisms, from prokaryotes to vertebrates. GABA is widely distributed in nonneural tissue including peripheral nervous and endocrine systems. GABA acts on GABAA and GABAB receptors. GABAA receptors are ligand-gated chloride channels modulated by a variety of drugs. GABAB receptors are essentially presynaptic, usually coupled to potassium or calcium channels, and they function via a GTP binding protein. In neural and nonneural tissues, GABA is metabolized by three enzymes--glutamic acid decarboxylase (GAD), which produces GABA from glutamic acid, and the catabolic enzymes GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Production of succinic acid by SSADH allows entry of the GABA carbon skeleton into the tricarboxylic acid cycle. Alternate sources of GABA include putrescine, spermine, spermidine and ornithine, which produce GABA via deamination and decarboxylation reactions, while L-glutamine is an additional source of glutamic acid via deamination. GAD from mammalian brain occurs in two molecular forms, GAD65 and GAD67 (referring to subunit relative molecular weight (Mr) in kilodaltons). These different forms of GAD are the product of different genes, differing in nucleotide sequence, immunoreactivity and subcellular localization. The presence and characteristics of GAD have been investigated in a wide variety of nonneural tissues including liver, kidney, pancreas, testis, ova, oviduct, adrenal, sympathetic ganglia, gastrointestinal tract and circulating erythrocytes. In some tissues, one form (GAD65 or GAD67) predominates. GABA-T has been located in most of the same tissues, primarily through histochemical and/or immunochemical methods; GABA-T is also present in a variety of circulating cells, including platelets and lymphocytes. SSADH, the final enzyme GABA catabolism, has been detected in some of the tissues in which GAD and GABA-T have been identified, although the presence of this enzyme has not been in mammalian pancreas, ova, oviduct, testis or sympathetic ganglia.

We recently reported that the mammalian brain has two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD, E.C. 4.1.1.15), which are the products of two genes. The two forms, which we call GAD65 and GAD67, differ from each other in sequence, molecular size, subcellular distribution, and interactions with the cofactor pyridoxal phosphate (PLP), with GAD65 activity more dependent than that of GAD67 on the continued presence of exogenous PLP. The existence of two GAD genes suggests that individual GABA neurons may be subject to differential regulation of GABA production. We have examined the expression of these two forms of GAD during postnatal development of the rat striatum to determine whether different classes of GABA neurons selectively express different amounts of the two GAD mRNAs. Here we present evidence for a dramatic developmental difference in the expression of the two mRNAs during postnatal development of the rat striatum. Using in situ hybridization to the two GAD mRNAs, we observed a selective increase in GAD65 mRNA during the second postnatal week, at the time when striatal matrix neurons innervate the substantia nigra (SN). PLP-dependent enzyme activity in the midbrain increases in parallel with increased expression of GAD65 mRNA in the striatum. We hypothesize that the innervation of the SN by striatal neurons triggers an increase in GAD65. The changing ratios of GAD65 and GAD67 in the striatum may contribute to the well-documented changes in seizure susceptibility that occur in early life.

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD67, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD67 mRNA predominates in the cerebellum. The mRNA for GAD65, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD65 is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD67 mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.

The enzyme glutamic acid decarboxylase (GAD, E.C. 4.1.1.15) is responsible for the biosynthesis of GABA, one of the most abundant inhibitory neurotransmitters in the vertebrate brain. In humans, pathophysiology involving GABA inhibitory systems can lead to movement disorders, seizures, and mental dysfunction (1–3). Detection of GAD mRNA by in situ hybridization can allow new approaches to the study of the regulation of these important systems.

Glutamic acid decarboxylase (GAD) mRNA was quantified in different regions of rat brain using an antisense RNA probe (ribo-probe) prepared from a cloned feline cDNA. In all brain regions studied a single band of GAD mRNA of approximately 3.7 kb was detected. The level of GAD mRNA was highest in the cerebellum, followed by the hypothalamus greater than thalamus greater than striatum greater than hippocampus greater than frontal cortex = parietal cortex greater than or equal to medulla = pons. Since GAD has been previously localized to intrinsic neurons of the striatum, we examined the effects of intrastriatal kainic acid administration on striatal GAD mRNA. The level of GAD mRNA in the kainic acid-lesioned striatum was reduced by 70-75% when compared to the contralateral (unlesioned) striatum. In contrast, the level of glutamine synthetase (an enzyme localized to glia) mRNA was increased approximately 290% in the kainic acid-lesioned striatum. There were no significant differences in GAD mRNA levels between the ipsilateral and contralateral cerebral cortices and hippocampi of rats injected with intrastriatal kainic acid.