Altered Calcitonin Gene-related Peptide, Substance P and Enkephalin Immunoreactivities and Receptor Binding Sites in the Dorsal Spinal Cord of the Polyarthritic Rat

The dorsal horn of the spinal cord, which forms the locus of first synapses in pain pathways, is an important site of interaction between calcitonin gene-related peptide (CGRP), substance P and enkephalin—the neuropeptides considered to be especially involved in the regulation of pain perception. Since adjuvant-induced arthritic rats provide a suitable model for peripheral inflammation and hyperalgesia, the possible alterations of immunoreactive CGRP, substance P and enkephalin as well as the binding sites for [¹²⁵I]hCGRPα, [¹²⁵I]substance Plneurokinin-1, (NK1) and [¹²⁵I]FK-33-824/μ-opioid receptors were studied in the dorsal horn of the spinal cord receiving projections from the inflamed limbs. In arthritic rats compared to control animals, a bilateral increase in CGRP- and substance P-immunoreactive fibres and the presence of enkephalin-immunoreactive cell bodies were noted in the dorsal horn of the spinal cord. As for receptors, while a significant decrease in [¹²⁵I]hCGRPα and [¹²⁵I]substance P/NK1 binding sites was observed in selective layers, no measurable alteration in [¹²⁵I]FK-33-824/μ-opioid binding sites was noted in any regions of the arthritic rat dorsal horn compared to the unaffected control rats. Following unilateral section of the peripheral nerve prior to induction of arthritis, CGRP- and substance P-immunoreactive fibres were markedly depleted and no enkephalin-positive neurons were observed in the ipsilateral dorsal horn. Analysis of receptor binding sites in denervated arthritic rats, however, exhibited differential responses, i.e. a significant increase in [¹²⁵I]hCGRPα, a marked decrease in [¹²⁵I]FK-33-824/μ-opioid and apparently no alteration in [¹²⁵I]substance P/NK1 receptor binding sites were observed in the ipsilateral dorsal horn compared to the intact contralateral side. These results taken together provide anatomical evidence for a concerted role of these peptides in the regulation of adjuvant-induced hyperalgesia accompanying peripheral inflammation.     

Flip and Flop Variants of AMPA Receptors in the Rat Lumbar Spinal Cord

The expression of eight messenger RNA splice forms encoding the Flip and Flop variants of AMPA receptor subunits GluR-A to -D in the rat lumbar spinal cord was examined by in situ hybridization using specific oligonucleotides. In the dorsal horn (laminae I, II and III) the predominant mRNA was GluR-B Flip. Much lower levels of GluR-A Flip were found in lamina I and in superficial parts of lamina II outer. In the ventral horn, motor neurons expressed mainly GluR-B Flip, GluR-C Flip and Flop, and GluR-D Flip. Serial sectioning through large motor neurons indicated that a given cell contained, for example, both GluR-C Flip and Flop splice types.     

Synaptic Activation of GABAA Receptors Causes a Depolarizing Potential Under Physiological Conditions in Rat Hippocampal Pyramidal Cells

Intracellular recordings with K-acetate-filled microelectrodes were performed in slices of the adult rat hippocampus maintained in vitro at 35 - 36 degrees C to analyse the potentials associated with the orthodromic inhibitory sequence generated by CA1 pyramidal cells. In 43 of 72 cells, stimuli that were delivered in the stratum radiatum induced (i) an initial excitatory postsynaptic potential (EPSP), (ii) an early, hyperpolarizing inhibitory postsynaptic potential (IPSP) (peak latency from the stimulus artefact 20 ms), (iii) an intermediate depolarizing component (peak latency=60 - 120 ms; duration=60 - 150 ms, and (iv) a late, long-lasting hyperpolarizing IPSP (peak latency=120 - 160 ms, duration >400 ms). In the remaining cells the orthodromic inhibitory response lacked the intermediate depolarization. The depolarizing component was selectively blocked by local applications of bicuculline or picrotoxin on the apical dendrites of pyramidal cells. This pharmacological procedure induced an increase in the amplitude of the EPSP that was capable of triggering 2 - 3 action potentials, but no reduction of the recurrent IPSP which is caused by GABAA receptors located close to the soma. The amplitude and duration of the depolarizing component was enhanced by lowering the temperature in the tissue chamber to 29 - 31 degrees C or by application of the GABA uptake blocker nipecotic acid, further indicating that the depolarizing component represented an active phenomenon mediated through GABA. Application of the Cl- pump blocker furosemide reduced and eventually blocked the early IPSP and the depolarizing component. These data demonstrate that under physiological conditions rat hippocampal pyramidal cells generate a depolarization that is presumably caused by an outwardly directed Cl- movement due to the activation of GABAA receptors located on the apical dendrites. This novel mechanism might modulate hippocampal excitability in both physiological and pathophysiological conditions.     

GABA Levels in Cerebrospinal Fluid of Patients with Epilepsy

Abstract: The lumbar cerebrospinal fluid (CSF) γ-aminobutyric acid (GABA) levels were measured in 27 patients with epilepsy, another three epileptic patients with status epilepticus and three epileptic patients with chronic cerebellar ataxia. The mean lumbar CSF GABA levels of the 27 patients with epilepsy were not significantly different from those of normal controls. Six of these 27 patients who had daily partial complex and partial motor seizures showed significantly low CSF GABA levels as did the six other patients, three each with status epilepticus and chronic cerebellar ataxia. These findings suggest that some epileptic patients have impaired brain GABAergic neurons.     

Localization of Dopamine D2 Receptor in Rat Spinal Cord Identified with Immunocytochemistry and In Situ Hybridization

In the present study the distribution of dopamine D₂ receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D₂ receptor and in situ hybridization (ISH) using a [³⁵S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D₂ receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D₂ receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D₂ receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D₂ receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.     

Actions of Somatostatin on Rat Neocortical Neurons in vitro

The effects of somatostatin (SS14) on neocortical neurons of the rat were investigated in an in vitro slice preparation. Intracellular recordings were performed in neurons (n=30) in layers 2 and 3 of the frontal cortex. Iontophoretically applied SS14 reduced the responses evoked by iontophoretically applied L-glutamate (GLU) and gamma-aminobutyric acid (GABA). The blocking effect of SS14 was apparent 1 - 2 min after onset of SS14 application and recovery required 2 - 3 min. The conductance increase evoked by GLU or GABA was reduced by SS14. In the majority of neurons, SS14 did not produce any measurable changes in passive membrane properties, spike threshold or on orthodromically evoked synaptic potentials. In 5 cells, SS14 induced a slight hyperpolarization (<3 mV). These results lend further support to claims that SS14 plays a neuromodulatory role in the neocortex.     

GABAergic neurosteroids mediate the effects of ethanol on long-term potentiation in rat hippocampal slices

We previously found that ethanol has complex effects on hippocampal synaptic plasticity, inhibiting long-term potentiation (LTP) and long-term depression by different mechanisms. The block of long-term depression appears to be mediated by effects on N-methyl-d-aspartate receptors, whereas the block of LTP involves augmented inhibition via gamma-aminobutyric acid-A receptors (GABA(A)Rs). To pursue factors contributing to effects on LTP, we examined the ability of various concentrations of ethanol to block LTP in the CA1 region of rat hippocampal slices. Complete LTP block required 60 mm ethanol. LTP block was enhanced at lower ethanol concentrations in the presence of (3alpha5alpha)-3-hydroxypregnan-20-one, a GABA(A)R-potentiating neurosteroid, suggesting that neurosteroids may be important contributors to the effects of ethanol on LTP. Consistent with this, we found that block of LTP by 60 mm ethanol was overcome by coadministration of a cyclodextrin that binds and removes lipophilic neurosteroids. More specifically, treatment of slices with finasteride, an agent that inhibits the synthesis of 5alpha-reduced neurosteroids, or with an agent that inhibits the effects of 5alpha-reduced neurosteroids on GABA(A)Rs overcame the effects of 60 mm ethanol on LTP. Taken together, these results indicate that acute production of GABA(A)R-enhancing neurosteroids plays a key role in mediating the effects of ethanol on LTP.     

Membrane Characteristics and Synaptic Responsiveness of Superficial Dorsal Horn Neurons in a Slice Preparation of Adult Rat Spinal Cord

Intracellular recordings have been made from neurons of the superficial dorsal horn in slices of the lumbar and thoracic spinal cord of young adult rats. Three broad categories of neurons could be distinguished on the basis of their firing patterns to intracellular current pulses and their afterhyperpolarizations (AHP); there was no detectable difference in the regional distribution of the three types. Category 1 cells were characterized by maintained firing to intracellular depolarizing current pulses, brief action potential durations and polyphasic AHPs. Category 2 cells showed spike adaptation, without spike attenuation, during intracellular current pulses, and had monophasic AHPs. Category 3 cells fired only 1 or 2 spikes to maintained depolarizing pulses and had smaller monophasic AHPs than category 2 neurons. Spontaneous excitatory and inhibitory postsynaptic potential (epsp and ipsp) activity was seen with psp durations varying widely. Low intensity electrical stimulation of afferent fibres, or of superficial white matter, resulted in polyphasic epsps and/or ipsps. The spike discharge in response to such afferent inputs correlated with the membrane properties of the cells, such that the synaptic responses of category 1 neurons were usually bursts of spikes, whereas category 2 and 3 neurons either failed to fire or fired only a single spike. These results in adult rat spinal cord suggest that the discharge pattern within synaptic sensory responses of superficial dorsal horn neurons is determined by postsynaptic membrane properties as well as by the pattern of the afferent input.     

Localization of Oxytocin Binding Sites in the Thoracic and Upper Lumbar Spinal Cord of the Adult and Postnatal Rat: A Histoautoradiographic Study

Oxytocin binding sites were detected by autoradiography on films and emulsion-coated sections in the spinal cord of adult and postnatal rats from C8 to L2, using a highly selective ¹²⁵l-labelled oxytocin antagonist. Oxytocin binding sites were detected on all transverse sections in the dorsal horn, where labelling was scattered over laminae I and II. The autonomic areas, i.e. the intermediolateral cell column, the central grey (lamina X) and the nucleus intercalatus were labelled. Binding in the intermediolateral cell column was most frequently observed on sections from T9 to T11 in adult and T7 to T8 in postnatal rats. In this location, oxytocin binding sites were highly concentrated on cell bodies of putative sympathetic preganglionic neurons; however, not all of these cells were labelled. Diffuse labelling occurred on the dorsal part of the central grey, mainly between T8 and L2. Isolated labelled cells belonging to the nucleus intercalatus were scattered between the central canal and the intermediolateral cell column. In addition, oxytocin binding sites were found on some motoneurons of the lateral group of T12-T13, but only in postnatal rats. The distribution of oxytocin binding sites in the rat spinal cord coincides with that of the oxytocin innervation and strongly suggests a modulatory role of this peptide in sensory and autonomic functions.     

NMDA-Dependent GABAA-Mediated Polysynaptic Potentials in the Neonatal Rat Hippocampal CA3 Region

Evoked inhibitory postsynaptic potentials (IPSPs) were studied in CA3 hippocampal neurons from brain slice preparations of rats ranging from 5 to 18 days of age (P5–18) using intracellular recording techniques. With KMeSO₄-filled electrodes the evoked inhibitory response consisted of fast and slow IPSPs mediated by GABA_A and GABA_B receptors respectively. In recordings obtained with electrodes filled with 2-(triethylamino)- N -(2,6-dimethylphenyl) acetamide and KMeSO₄, electrical stimulation evoked monophasic IPSPs in mature slices (P10–18) and biphasic IPSPs with an early and a late phase in neonatal slices (P4–7). In neonates both the early and late phases of the IPSP were mediated by GABA_A receptors. Pharmacological investigation revealed that the early phase arose from both direct and feedforward activation of GABAergic interneurons involving non-NMDA receptors, while the late phase resulted from polysynaptic activation of GABAergic interneurons mediated by NMDA receptors.     

Cholecystokinin in Mammalian Primary Sensory Neurons and Spinal Cord: In Situ Hybridization Studies in Rat and Monkey

The peptide cholecystokinin (CCK) has been suggested to be involved in nociception, but its exact localization at the level of the spinal cord and in spinal ganglia has been a controversial issue. Therefore the distribution of messenger RNA (mRNA) for CCK was studied by in situ hybridization using oligonucleotide probes on sections of adult rat lumbar dorsal root ganglia following unilateral section of the sciatic nerve and on sections of untreated monkey trigeminal ganglia, spinal cord and spinal ganglia from all levels. For comparison, calcitonin gene-related peptide (CGRP) mRNA was also studied in the monkey tissue using the same techniques. Peripheral sectioning of the sciatic nerve in the rat resulted in the appearance of detectable CCK mRNA in up to 30% of remaining ipsilateral L4 and L5 dorsal root ganglion neurons 3 weeks after surgery, with a distinct but more limited appearance also in the contralateral ganglia. No cells, or only single cells, could be seen in normal control rat ganglia. In contrast, in the normal monkey, ∼20% of dorsal root ganglion neurons, regardless of spinal level, and 10% of trigeminal ganglia neurons expressed mRNA for CCK. CGRP mRNA was expressed at detectable levels in ∼80% of these monkey dorsal root ganglion neurons. In the monkey spinal cord, CCK mRNA was detected in the dorsal horn and in motoneurons, whereas CGRP mRNA was only seen in motoneurons. The present results suggest that CCK peptides can be involved in sensory processing in the dorsal horn of the spinal cord in normal monkeys and in rats after peripheral nerve injury, adding one more possible excitatory peptide to the group of mediators in the dorsal horn.     

The Neuroactive Steroid 5α-Tetrahydrodeoxycorticosterone Increases GABAergic Postsynaptic Inhibition in Rat Neocortical Neurons in vitro

The neuroactive steroid 5α-pregnane-3α,21-diol-20-one (5α-tetrahydrodeoxycorticosterone; 5α-THDOC) has been shown to potentiate GABA-induced chloride currents in cell cultures and subcellular preparations. In this study, we recorded from pyramidal neurons in an in vitro slice preparation of the adult rat frontal neocortex using intracellular microelectrodes. 5α-THDOC (10 μM) increased and prolonged the inhibitory postsynaptic potential (IPSP). The mean maximal synaptic conductance of the early, GABA_A receptor-mediated, IPSP was enhanced to more than 700%, the one at the maximum of the late, partially GABA_A receptor-mediated, IPSP to approximately 400%. The progesterone/glucocorticoid receptor antagonist RU 38486 did not prevent the IPSP increase. At a concentration of 1 μM 5α-THDOC increased only the early IPSP to about 125%. Responses to the iontophoretically applied specific GABA_A receptor agonist muscimol but not to the specific GABA_B receptor agonist L-baclofen were enhanced by 5α-THDOC (10 μM). In the giga-seal whole-cell configuration when the GABA_B receptor-mediated IPSP component was absent due to intracellular perfusion, 5α-THDOC (10 μM) increased IPSPs to a similar extent as in the conventional microelectrode recordings. Excitatory postsynaptic potentials, resting membrane potential, input resistance and action potential amplitude were not affected by 5α-THDOC (10 μM). These data demonstrate that in neocortical tissue of the rat 5α-THDOC enhances GABAergic inhibition by interacting with postsynaptic GABA_A receptors while synaptic excitation and parameters of electric excitability remain unchanged.     

Gamma-Aminobutyric Acid Modulation of Corticotrophin-Releasing Factor-41 Secretion from the Rat Hypothalamus in vitro

There is increasing evidence for a centrally mediated inhibitory effect of the amino-acid neurotransmitter y-aminobutyric acid (GABA) on the hypothalamo-pituitary-adrenal axis. In the present study, the direct effect of GABA in modulating the release of the 41-residue corticotrophin-releasing factor (CRF-41), the major CRF identified so far, was investigated in acute hypothalamic explants by utilizing previously validated incubation and assay techniques. While GABA (10^-7’to 10^-5 M) had no effect on basal CRF-41 release (P > 0.05), it significantly suppressed K ^- (28 mM)-stimulated release in a dose-dependent manner (P < 0.01). A similar inhibitory effect was observed with the GABA agonist muscimol (10^-7 to 10^-5 M). Noradrenaline (10^-6 M) -induced CRF-41 release was also significantly inhibited by GABA 10^-6 M. The inhibitory effect of GABA on K⁺-stimulated CRF-41 secretion was completely. reversed by the GABA antagonists bicuculline and picrotoxin (10^-6 to 10^-5 M) in a dose-dependent fashion. Both bicuculline and picrotoxin stimulated basal and K⁺ (28 mM)-stimulated CRF-41 release, indicating the presence of tonic inhibition by endogenous GABA in the basal state. Finally, GABA 10^-5 M was able to significantly inhibit the stimulated release of CRF-41 from the isolated median eminence. In summary, the present data provide strong evidence that GABA-induced inhibition of the hypothalamo-pituitary-adrenal axis is mediated, at least in part, through an inhibitory action on CRF-41 secretion. It is likely that these GABA receptors are located directly on CRF-41 neurons, probably on nerve terminals in the median eminence.     

Epidural Spinal Cord Stimulation: Anatomical and Electrical Properties of the Intraspinal Structures Relevant to Spinal Cord Stimulation and Clinical Correlations

Epidural spinal cord stimulation (SCS) has gained a secure place in the armamentarium of the surgeon treating chronic pain conditions 1 , 2 . The complexity of the intraspinal structures and their different susceptibility to electrical signals, however, has made it difficult to characterize the effects of the stimulation, Some important recent work has helped shed light on the electrical properties of the intraspinal structures and on the electrical field potentials generated with epidural spinal cord stimulation. This work, initially pioneered by Sin and Coburn, has successfully been expanded and perfected by Holsheimer and Strujik at the University of Twente, The Netherlands ( 3 - 8 ). The Dutch scientists developed a computerized volume conductor model of the spinal cord to represent in extreme detail the electrical properties of all the intraspinal structures, including the dorsal column and dorsal root fibers. The model can simulate the effects of epidural stimulation with different electrode geometries and configurations 8 . The data generated from the model were then validated by comparing them to a large number of data collected by the author in implanted subjects ( 9 - 12 ). The author also conducted a detailed analysis of the clinical properties of the activation of the intraspinal structures at various electrode positions in the spine 13 , 14 .     

GABAA Receptor Function in the Early Period After Transient Forebrain Ischaemia in the Rat

The purpose of this study was to evaluate the function of the GABA_A receptor following transient forebrain ischaemia. The GABA-stimulated chloride (³⁶Cl^?) uptake into synaptoneurosomes was determined as an indicator of GABA_A receptor function. Synaptoneurosomes were isolated from control rats and rats in which the forebrain was made ischaemic by way of the two-vessel occlusion model. Animals subjected to ischaemia were killed at the end of the ischaemic insult and at 30 min or 2 or 5 h of recirculation. The results showed a reduction of 75% in GABA-mediated ³⁶Cl^? uptake in synaptoneurosomes isolated from animals shortly (<0.5 h) after the ischaemic episode (P < 0.01). After longer recirculation periods the GABA-mediated ³⁶Cl^? uptake reached preischaemic control levels. To investigate whether alterations in ³⁶Cl^? uptake were related to the synaptoneurosomal metabolic status, the synaptoneurosomal ATP content was measured. The time course of the ATP recovery correlated with the recovery of the GABA-mediated ³⁶Cl^? uptake (r= 0.7, P < 0.001). To investigate the importance of ATP in GABA-mediated ³⁶Cl^?uptake more directly, synaptoneurosomes isolated from control rats were exposed to chemically induced ATP depletion with rotenone, an inhibitor of oxidative phosphorylation. This resulted in similar reductions in both ATP level and GABA-stimulated ³⁶Cl^? uptake as observed after in vivo ischaemia. These findings indicate that GABA_A receptor function is transiently impaired in the early postischaemic period in a way which is closely related to alterations in cellular energy metabolism. The relevance of these findings to the development of ischaemic cell death is discussed.