Proliferative activity and motoneurone recruitment persist at the spinal cord central canal during larval and some postlarval stages in the rainbow trout (Oncorhynchus mykiss)

We previously found a linear relationship between the cross sectional myotomal area and the motoneurone number in the growing trout during postlarval stages. These neurones increased in number until a fish length of 150 mm, which prompted us to examine how motor neurones are recruited afterwards to meet the growth of their target myotomal muscle. Young adult (260 mm in length), fingerlings (F, 120-170 mm), fry (Fr, 70 mm) and eleutherembryos (Es, 20-30 mm) of rainbow trout (Oncorhyncus mykiss) were employed in this study. PCNA immunohistochemistry was used for monitoring the proliferative activity in the epithelium of the spinal cord central canal. This activity was quantified as the number of PCNA labelled cells for each spinal cord section. In Es and Fry, a mean value of 3-5 labelled cells for each section was found with a sharp decrease in young F (120 mm long). After this fish length, it was not possible to quantitatively evaluate the proliferative activity at the central canal. However, labelled cells were seldom found in the spinal cord sections until a fish length of 260 mm. From these data it is possible to conclude that motoneurone recruitment in the trout spinal cord is down-regulated at the F stage. Afterwards, we found that motoneurones increase in size to meet the growth of their target myotomal muscle.     

Group I mGluR regulates the polarity of spike-timing dependent plasticity in substantia gelatinosa neurons

The spinal synaptic plasticity is associated with a central sensitization of nociceptive input, which accounts for the generation of hyperalgesia in chronic pain. However, how group I metabotropic glutamate receptors (mGluRs) may operate spinal plasticity remains essentially unexplored. Here, we have identified spike-timing dependent synaptic plasticity in substantia gelatinosa (SG) neurons, using perforated patch-clamp recordings of SG neuron in a spinal cord slice preparation. In the presence of bicuculline and strychnine, long-term potentiation (LTP) was blocked by AP-5 and Ca2+ chelator BAPTA-AM. The group I mGluR antagonist AIDA, PLC inhibitor U-73122, and IP3 receptor blocker 2-APB shifted LTP to long-term depression (LTD) without affecting acute synaptic transmission. These findings provide a link between postsynaptic group I mGluR/PLC/IP3-gated Ca2+ store regulating the polarity of synaptic plasticity and spinal central sensitization.     

Strategies to repair lost sensory connections to the spinal cord

Failure of injured axons to regenerate in the central nervous system (CNS) is the main obstacle for repair of stroke and traumatic injuries to the spinal cord and sensory roots. This regeneration failure is high-lighted at the dorsal root transitional zone (DRTZ), the boundary between the peripheral (PNS) and central nervous system where sensory axons enter the spinal cord. Injured sensory axons regenerate in the PNS compartment of the dorsal root but are halted as soon as they reach the DRTZ. The failure of regenerating dorsal root axons to re-enter the mature spinal cord is a reflection of the generally nonpermissive nature of the CNS environment, in contrast to the regeneration supportive properties of the PNS. The dorsal root injury paradigm is therefore an attractive model for studying mechanisms underlying CNS regeneration failure in general and how to overcome the hostile CNS environment. Here we review the main lines that have been pursued to achieve growth of injured dorsal root axons into the spinal cord: (i) modifying the inhibitory nature of the DRTZ by breaking down or blocking the effect of growth repelling molecules, (ii) stimulate elongation of injured dorsal root axons by a prior conditioning lesion or administration of specific growth factors, (iii) implantation of olfactory ensheathing cells to provide a growth supportive cellular terrain at the DRTZ, and (iv) replacing the regeneration deficient adult dorsal root ganglion neurons with embryonic neurons or neural stem cells.     

Cannabinoid CB2 Receptors Regulate Central Sensitization and Pain Responses Associated with Osteoarthritis of the Knee Joint

Osteoarthritis (OA) of the joint is a prevalent disease accompanied by chronic, debilitating pain. Recent clinical evidence has demonstrated that central sensitization contributes to OA pain. An improved understanding of how OA joint pathology impacts upon the central processing of pain is crucial for the identification of novel analgesic targets/new therapeutic strategies.Inhibitory cannabinoid 2 (CB₂) receptors attenuate peripheral immune cell function and modulate central neuro-immune responses in models of neurodegeneration. Systemic administration of the CB₂ receptor agonist JWH133 attenuated OA-induced pain behaviour, and the changes in circulating pro- and anti-inflammatory cytokines exhibited in this model. Electrophysiological studies revealed that spinal administration of JWH133 inhibited noxious-evoked responses of spinal neurones in the model of OA pain, but not in control rats, indicating a novel spinal role of this target. We further demonstrate dynamic changes in spinal CB₂ receptor mRNA and protein expression in an OA pain model. The expression of CB₂ receptor protein by both neurones and microglia in the spinal cord was significantly increased in the model of OA. Hallmarks of central sensitization, significant spinal astrogliosis and increases in activity of metalloproteases MMP-2 and MMP-9 in the spinal cord were evident in the model of OA pain. Systemic administration of JWH133 attenuated these markers of central sensitization, providing a neurobiological basis for analgesic effects of the CB₂ receptor in this model of OA pain. Analysis of human spinal cord revealed a negative correlation between spinal cord CB₂ receptor mRNA and macroscopic knee chondropathy.These data provide new clinically relevant evidence that joint damage and spinal CB₂ receptor expression are correlated combined with converging pre-clinical evidence that activation of CB₂ receptors inhibits central sensitization and its contribution to the manifestation of chronic OA pain. These findings suggest that targeting CB₂ receptors may have therapeutic potential for treating OA pain.     

Spinal cord injuries: making the re-connection

Why is it that the skin and other tissues and organs can repair themselves, yet the spinal cord and brain cannot? Even more intriguingly, how is it that peripheral nerve damage may be repairable, yet central nervous system damage is not? Molecular answers to these questions could lead to therapies that would heal a damaged spinal cord.     

Cholinergic input is required during embryonic development to mediate proper assembly of spinal locomotor circuits

Rhythmic limb movements are controlled by pattern-generating neurons within the ventral spinal cord, but little is known about how these locomotor circuits are assembled during development. At early stages of embryogenesis, motor neurons are spontaneously active, releasing acetylcholine that triggers the depolarization of adjacent cells in the spinal cord. To investigate whether acetylcholine-driven activity is required for assembly of the central pattern-generating (CPG) circuit, we studied mice lacking the choline acetyltransferase (ChAT) enzyme. Our studies show that a rhythmically active spinal circuit forms in ChAT mutants, but the duration of each cycle period is elongated, and right-left and flexor-extensor coordination are abnormal. In contrast, blocking acetylcholine receptors after the locomotor network is wired does not affect right-left or flexor-extensor coordination. These findings suggest that the cholinergic neurotransmitter pathway is involved in configuring the CPG during a transient period of development.     

A comparison of microdistributions of taurine and cysteine sulphinate decarboxylase activity with those of GABA and L-glutamate decarboxylase activity in rat spinal cord and thalamus

Abstract— Distribution profiles of taurine and activity of cysteine sulphinate decarboxylase (CSD), the enzyme catalysing the formations of hypotaurine from cysteine sulphinate and of taurine from cysteate respectively, in the rat spinal cord and thalamus were studied in comparison with those of GABA and activity of l -glutamate decarboxylase (GAD), the rate limiting enzyme for GABA formation. In the spinal cord (L₂-L₃), it was found that taurine is fairly evenly distributed, whereas the activity of CSD is higher in the dorsal half of the spinal cord than in the ventral half. The highest CSD activity was found in the dorsal part of the dorsal horn. In the anterior part (A 5.4) of the thalamus, taurine and CSD activity were also distributed evenly and no areas having high taurine content and CSD activity were detected. In contrast with the even distributions of taurine and CSD activity, both GABA and GAD activity were distributed unevenly in the same CNS areas examined: The areas having high GABA content and GAD activity in the thalamus (A 5.4) coincided with the ventrolateral part of the ventral nucleus of thalamus (VM), entopeduncular nucleus (EP) and nucleus reuniens thalami (RE), whereas those in the spinal cord were found to be in the dorsal part of the dorsal horn and surrounding parts of the central canal, respectively. Considering a probable role of GABA in mammalian central nervous system (CNS) as an inhibitory neurotransmitter, it seems unlikely that taurine acts as an inhibitory neurotransmitter at least in the rat spinal cord and thalamus.     

How do genes regulate simple behaviours? Understanding how different neurons in the vertebrate spinal cord are genetically specified

Understanding how the vertebrate central nervous system develops and functions is a major goal of a large body of biological research. This research is driven both by intellectual curiosity about this amazing organ that coordinates our conscious and unconscious bodily processes, perceptions and actions and by the practical desire to develop effective treatments for people with spinal cord injuries or neurological diseases. In recent years, we have learnt an impressive amount about how the nerve cells that communicate with muscles, motoneurons, are made in a developing embryo and this knowledge has enabled researchers to grow motoneurons from stem cells. Building on the success of these studies, researchers have now started to unravel how most of the other nerve cells in the spinal cord are made and function. This review will describe what we currently know about spinal cord nerve cell development, concentrating on the largest category of nerve cells, which are called interneurons. I will then discuss how we can build and expand upon this knowledge base to elucidate the complete genetic programme that determines how different spinal cord nerve cells are made and connected up into neural circuits with particular functions.     

Differential roles of spinal protein kinases C and a in development of primary heat and mechanical hypersensitivity induced by subcutaneous bee venom chemical injury in the rat

It has been demonstrated that subcutaneous injection of bee venom (BV) can produce different types of pain and hypersensitivity including persistent spontaneous nociception (PSN), primary heat and mechanical hypersensitivity (hyperalgesia) and mirror-image heat (MIH) hypersensitivity in an individual animal, and the changes of spinal neurons are likely to be responsible for the production of these pain-related behaviors. In this study, we examined the roles of spinal protein kinase C (PKC) and protein kinase A (PKA) in the BV-induced different types of pain and hypersensitivity in conscious rats. We found that: (1). BV-induced primary heat hypersensitivity could be blocked by intrathecal pre- or posttreatment with a PKC inhibitor, chelerythrine chloride (CH), while a PKA inhibitor, N-(2-[P-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89), had no effect. (2). BV-induced primary mechanical hypersensitivity could be blocked by pre- or posttreatment with H89, whereas CH had no effect. (3). Both pre- and posttreatment with H89 produced suppressive effects on both induction and maintenance of the BV-induced PSN and MIH hypersensitivity. Based on the present findings, we proposed that spinal PKC might be activated during the central processes of primary heat hypersensitivity, while spinal PKA is likely to be involved in primary mechanical hypersensitivity induced by subcutaneous BV chemical injury. Taken together with our previous report however, spinal PKC and PKA are likely to be simultaneously involved in the central processes of both PSN and MIH hypersensitivity.     

A new transgenic reporter line reveals expression of protocadherin 9 at a cellular level within the zebrafish central nervous system

The wiring of neuronal networks is far from understood. One outstanding question is how neurons of different types link up to form subnetworks within the greater context. Cadherins have been suggested to create an inclusion code where interconnected neurons express the same subtypes. Here, we have used a CRISPR/Cas9 knock-in approach to generate a transgenic zebrafish reporter line for protocadherin 9 (pcdh9), which is predominantly expressed within the central nervous system. Expression of eGFP was detected in subsets of neurons in the cerebellum, retina and spinal cord, in both larvae and juveniles. A closer characterization of the spinal locomotor network revealed that a portion of distinct classes of both excitatory and inhibitory interneurons, as well as motor neurons, expressed pcdh9. This transgenic line could thus be used to test the cadherin network hypothesis, through electrophysiological characterization of eGFP positive cells, to show if these are synaptically connected and form a discrete network within the spinal cord.     

Spinal Cord Regeneration in Amphibians: A Historical Perspective

In some vertebrates, a grave injury to the central nervous system (CNS) results in functional restoration, rather than in permanent incapacitation. Understanding how these animals mount a regenerative response by activating resident CNS stem cell populations is of critical importance in regenerative biology. Amphibians are of a particular interest in the field because the regenerative ability is present throughout life in urodele species, but in anuran species it is lost during development. Studying amphibians, who transition from a regenerative to a nonregenerative state, could give insight into the loss of ability to recover from CNS damage in mammals. Here, we highlight the current knowledge of spinal cord regeneration across vertebrates and identify commonalities and differences in spinal cord regeneration between amphibians.     

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.     

Autoradiographic distribution of 125I calcitonin gene-related peptide binding sites in the rat central nervous system

Using autoradiographic method and 125I-Tyro rat CGRP as a ligand, receptor binding sites were demonstrated in the rat central nervous system. Saturation studies and Scatchard analysis of CGRP-binding to slide mounted tissue sections containing primarily cerebellum showed a single class of receptors with a dissociation constant of 0.96 nM and a Bmax of 76.4 fmol/mg protein. 125I-Tyro rat CGRP binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the telencephalon (medial prefrontal, insular and outer layers of the temporal cortex, nucleus accumbens, fundus striatum, central and inferior lateral amygdaloid nuclei, most caudal caudate putamen, organum vasculosum laminae terminalis, subfornical organ), the diencephalon (anterior hypothalamic, suprachiasmatic, arcuate, paraventricular, dorsomedial, periventricular, reuniens, rhomboid, lateral thalamic pretectalis and habenula nuclei, zona incerta), in the mesencephalon (superficial layers of the superior colliculus, central nucleus of the geniculate body, inferior colliculus, nucleus of the fifth nerve, locus coeruleus, nucleus of the mesencephalic tract, the dorsal tegmental nucleus, superior olive), in the molecular layer of the cerebellum, in the medulla oblongata (inferior olive, nucleus tractus solitarii, nucleus commissuralis, nuclei of the tenth and twelfth nerves, the prepositus hypoglossal and the gracilis nuclei, dorsomedial part of the spinal trigeminal tract), in the dorsal gray matter of the spinal cord (laminae I-VI) and the confines of the central canal. Moderate receptor densities were found in the septal area, the "head" of the anterior caudate nucleus, medial amygdaloid and bed nucleus of the stria terminalis, the pyramidal layers of the hippocampus and dentate gyri, medial preoptic area, ventromedial nucleus, lateral hypothalamic and ventrolateral thalamic area, central gray, reticular part of the substantia nigra, parvocellular reticular nucleus. Purkinje cell layer of the cerebellum, nucleus of the spinal trigeminal tract and gracile fasciculus of the spinal cord. The discrete distribution of CGRP-like binding sites in a variety of sensory systems of the brain and spinal cord as well as in thalamic and hypothalamic areas suggests a widespread involvement of CGRP in a variety of brain functions.     

In vivo imaging of the mouse spinal cord using two-photon microscopy

In vivo imaging using two-photon microscopy in mice that have been genetically engineered to express fluorescent proteins in specific cell types has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task. We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.     

Central Administration of C-X-C Chemokine Receptor Type 4 Antagonist Alleviates the Development and Maintenance of Peripheral Neuropathic Pain in Mice

Aim

To explore the roles of C-X-C chemokine receptor type 4 (CXCR4) in spinal processing of neuropathic pain at the central nervous system (CNS).

Methods

Peripheral neuropathic pain (PNP) induced by partial sciatic nerve ligation (pSNL) model was assessed in mice. Effects of a single intrathecal (central) administration of AMD3100 (intrathecal AMD3100), a CXCR4 antagonist, on pain behavior and pain-related spinal pathways and molecules in the L3-L5 spinal cord segment was studied compare to saline treatment.

Results

Rotarod test showed that intrathecal AMD3100 did not impair mice motor function. In pSNL-induced mice, intrathecal AMD3100 delayed the development of mechanical allodynia and reversed the established mechanical allodynia in a dose-dependent way. Moreover, intrathecal AMD3100 downregulated the activation of JNK1 and p38 pathways and the protein expression of p65 as assessed by western blotting. Real-time PCR test also demonstrated that substance P mRNA was decreased, while adrenomedullin and intercellular adhesion molecule mRNA was increased following AMD3100 treatment.

Conclusion

Our results suggest that central (spinal) CXCR4 is involved in the development and maintenance of PNP and the regulation of multiple spinal molecular events under pain condition, implicating that CXCR4 would potentially be a therapeutic target for chronic neuropathic pain.     

Spatiotemporal Expression of Dexras1 After Spinal Cord Transection in Rats

Dexras1, a brain-enriched member of the Ras subfamily of GTPases, as a novel physiologic nitric oxide (NO) effector, anchor neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI), to specific targets to enhance NO signaling, and is strongly and rapidly induced during treatment with dexamethasone. It is unknown how the central nervous system (CNS) trauma affects the expression of Dexras1. Here we used spinal cord transection (SCT) model to detect expression of Dexras1 at mRNA and protein level in spinal cord homogenates by real-time PCR and Western blot analysis. The results showed that Dexras1 mRNA upregulated at 3 day, 5 day, and 7 day significantly (P < 0.05) that was consistent with the protein level except at 7 day. Immunofluorescence revealed that both neurons and glial cells showed Dexras1 immunoreactivivty (IR) around SCT site, but the proportion is different. Importantly, injury-induced expression of Dexras1 was co-labeled by caspase-3 (apoptotic marker) and Tau-1 (marker for pathological oligodendrocyte). Furthermore, colocalization of Dexras1, carboxy-terminal PSD95/DLG/ZO-1 (PDZ) ligand of nNOS (CAPON) and nNOS was observed in neurons and glial cells, supporting the existence of ternary complexes in this model. Thus, the results that the transient high expression of Dexras1 which localized in apoptotic neurons and pathological oligodendrocytes might provide new insight into the secondary response after SCT. Xin Li, Chun Cheng, and Min Fei contributed equally to this work.     

Characterization of central nervous system structures by magnetic resonance diffusion anisotropy

Diffusion-weighted magnetic resonance imaging (MRI) provides information about tissue water diffusion. Diffusion anisotropy, which can be measured with diffusion tensor MRI, is a quantitative measure of the directional dependence of the diffusion restriction that is introduced by biological structures such as nerve fibers. Diffusion tensor MRI data was obtained in the brain, brain stem, and cervical spinal cord. For each region, scans were performed in four normal volunteers. Fractional anisotropy (FA), an index of diffusion anisotropy, was measured within regions of interest located in the corpus callosum, capsula interna, thalamus, caudate nucleus, putamen, brain cortex, pyramidal tract of the medulla, accessory olivary nucleus, dorsal olivary nucleus, inferior olivary nucleus, spinal white and gray matter. The highest FA value was measured in the corpus callosum (81 +/- 3%). The values of the other areas decreased in the following order: pyramidal tract in the medulla (72 +/- 1%), spinal white matter (65 +/- 4%), capsula interna (62 +/- 3%), accessory olivary nucleus (36 +/- 2%), spinal gray matter (35 +/- 5%), dorsal olivary nucleus in the medulla (29 +/- 2%), thalamus (28 +/- 2%), inferior olivary nucleus (15 +/- 2%), putamen (13 +/- 2%), caudate nucleus (13 +/- 2%), and brain cortex (9 +/- 1%). Our results indicate that the underlying fiber architecture, fiber density, and uniformity of nerve fiber direction affect anisotropy values of the various structures. Characterization of various central nervous system structures with diffusion anisotropy is possible and may be useful to monitor degenerative diseases in the central nervous system.     

Spinal cord central canal of the German shepherd dog: Morphological,histological, and ultrastructural considerations

This study deals with some macroscopical, microscopical, and ultrastructural aspects of the spinal cord central canal of the German shepherd dog. The caudal end of the spinal cord is constituted by the conus medullaris, which may extend to the first sacral vertebra, the terminal ventricle, and the filum terminale. The latter structure is considered as internum (second to third sacral vertebrae) or externum (fifth caudal vertebra), according to its relation to the dura mater. Occasionally, there is a second anchorage which is close to the level of the sixth caudal vertebra. The central canal is surrounded by a ciliated ependymal epithelium, which differs depending upon the levels. The most caudal part of the filum terminale bears a columnar ciliated ependymal epithelium surrounded by two layers of glia and pia mater, which separate the central canal from the subarachnoid space. Microfil injections show a communication between the cavity and the subarachnoid space, as the plastic is able to pass through the ependymal epithelium. At the level of the terminal ventricle there are real separations of the ependymal epithelium, which seem to connect the lumen of the spinal canal with the subarachnoid space. These structures probably constitute one of the drainage pathways of the cerebrospinal fluid. The diameter of the central canal is related to the age of the animal. However, even in very old animals the spinal cord central canal reaches the tip of the filum terminale and remains patent until death. At the ultrastructural level the ependymal cells present villi, located on cytoplasmic projections, cilia, dense mitochondria, and oval nuclei. © 1995 Wiley-Liss, Inc.     

Increased expression of nitric oxide synthase interacting protein (NOSIP) following traumatic spinal cord injury in rats

Previous studies indicated that nitric oxide (NO) is involved in secondary damage of spinal cord injury (SCI), which worsens the primary physical injury to the central nervous systems. Recently, nitric oxide synthase interacting protein (NOSIP) has been identified to interact with neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase by inhibiting the NO production. However, its expression and function after a central nervous system injury remains unclear. In this study, we examined the expression and cellular localization of NOSIP in the spinal cord of an adult rat. Western blot analysis indicated that NOSIP protein levels increased at day1 post-injury and peaked at day 14. Double immunofluorescence staining showed that NOSIP was primarily expressed in neurons and glial cells in the intact spinal cord. Interestingly, this study also showed that the expression of NOSIP significantly increased in astrocytes after injury. Furthermore, injury-induced expression of NOSIP was co-expressed with proliferating cell nuclear antigen (PCNA) positive astrocytes after injury. We also showed the NOSIP was co-localized with nNOS in gray matter and white matter after SCI. All these data taken together suggested that NOSIP may play an important roles in astrogliogenesis after a spinal cord injury.     

CL 218,872 Binding to Benzodiazepine Receptors in Rat Spinal Cord: Modulation by γ-Aminobutyric Acid and Evidence for Receptor Heterogeneity

The binding of the triazolopyridazine CL 218,872 to central benzodiazepine receptors identified with [3H]Ro 15-1788 was studied in extensively washed homogenates of rat spinal cord and cerebral cortex. CL 218,872 displacement curves were shallow in both spinal cord (nH = 0.67) and cortex (nH = 0.54), suggesting the presence of type 1 and type 2 benzodiazepine receptors in both tissues. CL 218,872 had lower affinity in spinal cord (IC50 = 825 nM) than cortex (IC50 = 152 nM), possibly reflecting the presence of fewer type 1 sites in the cord. Activating gamma-aminobutyric acid (GABA) receptors with 10 microM muscimol resulted in a two- to threefold increase in CL 218,872 affinity in both tissues without changes in the displacement curve slope. This indicates that GABA enhances CL 218,872 affinity for both type 1 and type 2 sites in both spinal cord and cerebral cortex.