Inflammatory diseases and neuropathic insults are frequently accompanied by severe and debilitating pain, which can become chronic and often unresponsive to conventional analgesic treatment. A loss of synaptic inhibition in the spinal dorsal horn is considered to contribute significantly to this pain pathology. Facilitation of spinal gamma-aminobutyric acid (GABA)ergic neurotransmission through modulation of GABA(A) receptors should be able to compensate for this loss. With the use of GABA(A)-receptor point-mutated knock-in mice in which specific GABA(A) receptor subtypes have been selectively rendered insensitive to benzodiazepine-site ligands, we show here that pronounced analgesia can be achieved by specifically targeting spinal GABA(A) receptors containing the alpha2 and/or alpha3 subunits. We show that their selective activation by the non-sedative ('alpha1-sparing') benzodiazepine-site ligand L-838,417 (ref. 13) is highly effective against inflammatory and neuropathic pain yet devoid of unwanted sedation, motor impairment and tolerance development. L-838,417 not only diminished the nociceptive input to the brain but also reduced the activity of brain areas related to the associative-emotional components of pain, as shown by functional magnetic resonance imaging in rats. These results provide a rational basis for the development of subtype-selective GABAergic drugs for the treatment of chronic pain, which is often refractory to classical analgesics.

SUMMARY1. O2-,K+-and pH-sensitive microelectrodes were used to measure extracellular oxygen pressure (Po2), K' activity (aK.) and pH (pH.) in ventral regions of the medulla oblongata containing respiratory neurons in the in vitro brainstem-spinal cord preparation from 0 to 4-day-old rats.2. The location of respiratory neurons was mapped by extracellular recordings with conventional microelectrodes, or with the reference barrel of ion-sensitive microelectrodes. The major populations of respiratory neurons were distributed in the ventrolateral reticular formation near the nucleus ambiguus at depths of 300-600 ,um. In this area, aK. baseline increased from 3-2 to 3-8 mm whereas steadystate values of Po2 and pHo fell from 120 to 7 mmHg and from 6-9 to 6-7, respectively.3. During rhythmic inspiratory discharges recorded with suction electrodes from ventral roots of spinal (C3-C5) and cranial (IX, X, XII) nerves, aKo transiently increased by up to 100 /M, and Po2 fell maximally by 0 4 mmHg. During episodes of non-rhythmic neuronal discharge, aKo increased by as much as 0-4 mm and Po2 decreased by about 10 mmHg. In contrast, no variations in pHo could be detected during such activities.4. Activation of medullary neurons by tetanic electrical stimulation of axonal tracts in the ventrolateral column of the spinal cord at the level of the phrenic motoneuron pool produced aKo elevations of up to 5 mm, decreases of Po2 by up to 50 mmHg, and pHo increases by a maximum of 0 07 pH units. These aKo and Po2 transients were reduced by more than 80 % during blockade of synaptic transmission with 5 mm manganese (Mn2+) and completely blocked by 1 ,tm tetrodotoxin (TTX).5. The tissue Po2 gradient as well as activity-related decreases of P2 were completely abolished after block of oxidative cellular metabolism by addition of 2-10 mm cyanide (CN-) to the bathing solution.6. Inhibition of the Na+-K+ pump by addition of 3-50 /M ouabain (3-10 min)caused a reversible increase of aKo by 0-8-3 mm, a delayed recovery of stimulusinduced aKo elevations, and produced a disturbance of the respiratory rhythm.7. The sensitivity of the respiratory network to oxygen depletion was tested by superfusing the neuraxis with hypoxic solutions gassed with N2 instead of 02(5-20 min). The response of the respiratory network to such hypoxic exposure MS 9830 J. BROCKHA US AND OTHERS consisted of an initial increase in frequency of respiratory motor output, followed by a depression of respiratory activity that terminated in a reversible loss of the respiratory rhythm.8 10. It is concluded that the oxygen supply of the ventral respiratory network is sufficient to maintain aerobic neuronal metabolism and metabolically driven Na+-K+ pump activity which allows respiratory rhythmogenesis and network function in the in vitro neonatal rat brainstem-spinal cord preparation.

Interleukin-6 (IL-6) is a key mediator of inflammation. Inhibitors of IL-6 or of its signal transducing receptor gp130 constitute a novel class of anti-inflammatory drugs, which raise great hopes for improved treatments of painful inflammatory diseases such as rheumatoid arthritis. IL-6 and gp130 may enhance pain not only indirectly through their proinflammatory actions but also through a direct action on nociceptors (i.e., on neurons activated by painful stimuli). We found indeed that the IL-6/gp130 ligand-receptor complex induced heat hypersensitivity both in vitro and in vivo. This process was mediated by activation of PKC-␦ via Gab1/2/PI 3 K and subsequent regulation of TRPV1, a member of the transient receptor potential (TRP) family of ion channels. To assess the relevance of this direct pain promoting effect of IL-6, we generated conditional knock-out mice, which lack gp130 specifically in nociceptors, and tested them in models of inflammatory and tumor-induced pain. These mice showed significantly reduced levels of inflammatory and tumor-induced pain but no changes in immune reactions or tumor growth. Our results uncover the significance of gp130 expressed in peripheral pain sensing neurons in the pathophysiology of major clinical pain disorders and suggest their use as novel pain relieving agents in inflammatory and tumor pain.

Gramicidin-perforated patch-clamp recording revealed phasic Cl(-)-mediated hyperpolarizations in respiratory neurons of the brainstem-spinal cord preparation from newborn rats. The in vitro respiratory rhythm persisted after block of gamma-aminobutyric acid (GABA), i.e. GABAA, receptor-mediated inhibitory postsynaptic potentials (IPSPs) with bicuculline and/or glycinergic IPSPs with strychnine. In one class of expiratory neurons, bicuculline unmasked inspiration-related excitatory postsynaptic potentials (EPSPs), leading to spike discharge. Bicuculline also blocked hyperpolarizations and respiratory arrest due to bath-applied muscimol, whereas strychnine antagonized similar responses to glycine. The reversal potential of respiration-related IPSPs and responses to GABA, muscimol or glycine was not affected by CO2/HCO3(-)-free solutions, but shifted from about -65 mV to values more positive than -20 mV upon dialysis of the cells with 144 instead of 4 mM Cl-. Impairment of GABA uptake with nipecotic acid or glycine uptake with sarcosine evoked a bicuculline- or strychnine-sensitive decrease of respiratory frequency which could lead to respiratory arrest. Also, the GABAB receptor agonist baclofen led to reversible suppression of respiratory rhythm. This in vitro apnoea was accompanied by a K+ channel-mediated hyperpolarization (reversal potential -88 mV) of tonic cells, whereas membrane potential of neighbouring respiratory neurons remained almost unaffected. Both baclofen-induced hyperpolarization and respiratory depression were antagonised by 2-OH-saclofen, which did not affect respiration-related IPSPs per se. The results show that synaptic inhibition is not essential for rhythmogenesis in the isolated neonatal respiratory network, although (endogenous) GABA and glycine have a strong modulatory action. Hyperpolarizing IPSPs mediated by GABAA and glycine receptors provide a characteristic pattern of membrane potential oscillations in respiratory neurons, whereas GABAB receptors rather appear to be a feature of non-respiratory neurons, possibly providing excitatory drive to the network.

Action potential-evoked neurotransmitter release is impaired in knock-out neurons lacking synaptic cell-adhesion molecules α-neurexins (αNrxns), the extracellularly longer variants of the three vertebrate genes. Ca influx through presynaptic high-voltage gated calcium channels like the ubiquitous P/Q-type (Ca2.1) triggers release of fusion-ready vesicles at many boutons. α2δ Auxiliary subunits regulate trafficking and kinetic properties of Ca2.1 pore-forming subunits but it has remained unclear if this involves αNrxns. Using live cell imaging with Ca indicators, we report here that the total presynaptic Ca influx in primary hippocampal neurons of αNrxn triple knock-out mice of both sexes is reduced and involved lower Ca2.1-mediated transients. This defect is accompanied by lower vesicle release, reduced synaptic abundance of Ca2.1 pore-forming subunits, and elevated surface mobility of α2δ-1 on axons. Overexpression of Nrxn1α in αNrxn triple knock-out neurons is sufficient to restore normal presynaptic Ca influx and synaptic vesicle release. Moreover, coexpression of Nrxn1α together with α2δ-1 subunits facilitates Ca influx further but causes little augmentation together with a different subunit, α2δ-3, suggesting remarkable specificity. Expression of defined recombinant Ca2.1 channels in heterologous cells validates and extends the findings from neurons. Whole-cell patch-clamp recordings show that Nrxn1α in combination with α2δ-1, but not with α2δ-3, facilitates Ca currents of recombinant Ca2.1 without altering channel kinetics. These results suggest that presynaptic Nrxn1α acts as a positive regulator of Ca influx through Ca2.1 channels containing α2δ-1 subunits. We propose that this regulation represents an important way for neurons to adjust synaptic strength. Synaptic transmission between neurons depends on the fusion of neurotransmitter-filled vesicles with the presynaptic membrane, which subsequently activates postsynaptic receptors. Influx of calcium ions into the presynaptic terminal is the key step to trigger vesicle release and involves different subtypes of voltage-gated calcium channels. We study the regulation of calcium channels by neurexins, a family of synaptic cell-adhesion molecules that are essential for many synapse properties. Using optical measurements of calcium influx in cultured neurons and electrophysiological recordings of calcium currents from recombinant channels, we show that a major neurexin variant facilitates calcium influx through P/Q-type channels by interacting with their α2δ-1 auxiliary subunits. These results propose a novel way how neurons can modulate the strength of distinct synapses.

A challenge in neuroscience is to understand the mechanisms underlying synapse formation. Most excitatory synapses in the brain are built on spines, which are actin-rich protrusions from dendrites. Spines are a major substrate of brain plasticity, and spine pathologies are observed in various mental illnesses. Here we investigate the role of neurobeachin (Nbea), a multidomain protein previously linked to cases of autism, in synaptogenesis. We show that deletion of Nbea leads to reduced numbers of spinous synapses in cultured neurons from complete knockouts and in cortical tissue from heterozygous mice, accompanied by altered miniature postsynaptic currents. In addition, excitatory synapses terminate mostly at dendritic shafts instead of spine heads in Nbea mutants, and actin becomes less enriched synaptically. As actin and synaptopodin, a spine-associated protein with actin-bundling activity, accumulate ectopically near the Golgi apparatus of mutant neurons, a role emerges for Nbea in trafficking important cargo to pre- and postsynaptic compartments.

Members of the Kir2 subfamily of inwardly rectifying K ϩ channels characterized by their strong current rectification are widely expressed both in the periphery and in the CNS in mammals. We have cloned from rat brain a fourth subfamily member, designated Kir2.4 (IRK4), which shares 53-63% similarity to Kir2.1, Kir2.2, or Kir2.3 on the amino acid level. In situ hybridization analysis identifies Kir2.4 as the most restricted of all Kir subunits in the brain. Kir2.4 transcripts are expressed predominantly in motoneurons of cranial nerve motor nuclei within the general somatic and special visceral motor cell column and thus are uniquely related to a functional system. Heterologous expression of Kir2.4 in Xenopus oocytes and mammalian cells gives rise to low-conductance channels (15 pS), with an affinity to the channel blockers Ba 2ϩ (K i ϭ 390 M) and Cs ϩ (K i ϭ 8.06 mM) 30-50-fold lower than in other Kir channels. Low Ba 2ϩ sensitivity allows dissection of Kir2.4 currents from other Kir conductances in hypoglossal motoneurons (HMs) in rat brainstem slices. The finding that Ba 2ϩ -mediated block of Kir2.4 in HMs evokes tonic activity and increases the frequency of induced spike discharge indicates that Kir2.4 channels are of major importance in controlling excitability of motoneurons in situ.

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