Synaptic and extrasynaptic γ-aminobutyric acid type A receptor clusters in rat hippocampal cultures during development

We have simultaneously measured the expression of postsynaptic γ-aminobutyric acid type A (GABAA) receptor clusters and of presynaptic boutons in neonatal rat hippocampal cultures between days 1 and 30. GABAA receptors were labeled with antibodies recognizing the extracellular domains of β2/3 and γ2 subunits. Boutons were visualized by activity-dependent uptake of the styryl dye FM4-64, or by antibodies against the presynaptic vesicular protein SV2 or the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). GABAA receptor clusters could be seen in living neurons already 6 h after culturing, much before presynaptic markers could be identified in nerve terminals. The densities of receptor clusters that contained the β2/3 subunits were constant between days 10 and 30 in culture, whereas γ2 subunit-containing clusters fluctuated and reached a maximum on day 20. SV2 and GAD staining could be measured from day 2 onwards. Clustering of GAD in presynaptic terminals and FM4-64 uptake were observed only at day 5 and afterward. SV2 staining and FM4-64 uptake increased in parallel between days 5 and 20 and remained constant thereafter. GAD-stained boutons were fewer than those labeled with other, less specific, presynaptic stains. They reached a maximum on day 20 and fell again toward day 30. Double labeling of GABAA receptors and of presynaptic boutons in neurons during differentiation showed that, even after 30 days in culture, large fractions of GABAA receptor clusters containing β2/3 and/or γ2 subunits remained extrasynaptic.

Functional disorders of the sympathetic nervous system in mice lacking the α1B subunit (Cav 2.2) of N-type calcium channels

N-type voltage-dependent Ca2+ channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, we have generated mice genetically deficient in the α1B subunit (Cav 2.2). The α1B-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to ω-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, was markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation were dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice was nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction.

The spinal biology in humans and animals of pain states generated by persistent small afferent input

Behavioral models indicate that persistent small afferent input, as generated by tissue injury, results in a hyperalgesia at the site of injury and a tactile allodynia in areas adjacent to the injury site. Hyperalgesia reflects a sensitization of the peripheral terminal and a central facilitation evoked by the persistent small afferent input. The allodynia reflects a central sensitization. The spinal pharmacology of these pain states has been defined in the unanesthetized rat prepared with spinal catheters for injection and dialysis. After tissue injury, excitatory transmitters (e.g., glutamate and substance P) acting though N-methyl-d-aspartate (NMDA) and neurokinin 1 receptors initiate a cascade that evokes release of (i) NO, (ii) cyclooxygenase products, and (iii) activation of several kinases. Spinal dialysis show amino acid and prostanoid release after cutaneous injury. Spinal neurokinin 1, NMDA, and non-NMDA receptors enhance spinal prostaglandin E2 release. Spinal prostaglandins facilitate release of spinal amino acids and peptides. Activation by intrathecal injection of receptors on spinal C fiber terminals (μ,/∂ opiate, α2 adrenergic, neuropeptide Y) prevents release of primary afferent peptides and spinal amino acids and blocks acute and facilitated pain states. Conversely, consistent with their role in facilitated processing, NMDA, cyclooxygenase 2, and NO synthase inhibitors act to diminish only hyperalgesia. Importantly, spinal delivery of several of these agents diminishes human injury pain states. This efficacy emphasizes (i) the role of facilitated states in humans, (ii) shows the importance of spinal systems in human pain processing, and (iii) indicates that these preclinical mechanisms reflect processes that regulate the human pain experience.

Brain-derived neurotrophic factor is an endogenous modulator of nociceptive responses in the spinal cord

The primary sensory neurons that respond to noxious stimulation and project to the spinal cord are known to fall into two distinct groups: one sensitive to nerve growth factor and the other sensitive to glial cell-line-derived neurotrophic factor. There is currently considerable interest in the ways in which these factors may regulate nociceptor properties. Recently, however, it has emerged that another trophic factor—brain-derived neurotrophic factor (BDNF)—may play an important neuromodulatory role in the dorsal horn of the spinal cord. BDNF meets many of the criteria necessary to establish it as a neurotransmitter/neuromodulator in small-diameter nociceptive neurons. It is synthesized by these neurons and packaged in dense core vesicles in nociceptor terminals in the superficial dorsal horn. It is markedly up-regulated in inflammatory conditions in a nerve growth factor-dependent fashion. Postsynaptic cells in this region express receptors for BDNF. Spinal neurons show increased excitability to nociceptive inputs after treatment with exogenous BDNF. There are both electrophysiological and behavioral data showing that antagonism of BDNF at least partially prevents some aspects of central sensitization. Together, these findings suggest that BDNF may be released from primary sensory nociceptors with activity, particularly in some persistent pain states, and may then increase the excitability of rostrally projecting second-order systems. BDNF released from nociceptive terminals may thus contribute to the sensory abnormalities associated with some pathophysiological states, notably inflammatory conditions.

Transcriptional and posttranslational plasticity and the generation of inflammatory pain

Inflammatory pain manifests as spontaneous pain and pain hypersensitivity. Spontaneous pain reflects direct activation of specific receptors on nociceptor terminals by inflammatory mediators. Pain hypersensitivity is the consequence of early posttranslational changes, both in the peripheral terminals of the nociceptor and in dorsal horn neurons, as well as later transcription-dependent changes in effector genes, again in primary sensory and dorsal horn neurons. This inflammatory neuroplasticity is the consequence of a combination of activity-dependent changes in the neurons and specific signal molecules initiating particular signal-transduction pathways. These pathways phosphorylate membrane proteins, changing their function, and activate transcription factors, altering gene expression. Two distinct aspects of sensory neuron function are changed as a result of these processes, basal sensitivity, or the capacity of peripheral stimuli to evoke pain, and stimulus-evoked hypersensitivity, the capacity of certain inputs to generate prolonged alterations in the sensitivity of the system. Posttranslational changes largely alter basal sensitivity. Transcriptional changes both potentiate the system and alter neuronal phenotype. Potentiation occurs as a result of the up-regulation in the dorsal root ganglion of centrally acting neuromodulators and simultaneously in the dorsal horn of their receptors. This means that the response to subsequent inputs is augmented, particularly those that induce stimulus-induced hypersensitivity. Alterations in phenotype includes the acquisition by A fibers of neurochemical features typical of C fibers, enabling these fibers to induce stimulus-evoked hypersensitivity, something only C fiber inputs normally can do. Elucidation of the molecular mechanisms responsible provides new opportunities for therapeutic approaches to managing inflammatory pain.

Potentiation of capsaicin receptor activity by metabotropic ATP receptors as a possible mechanism for ATP-evoked pain and hyperalgesia

The capsaicin (vanilloid) receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been proposed that ATP, released from different cell types, initiates the sensation of pain by acting predominantly on nociceptive ionotropic purinoceptors located on sensory nerve terminals. In this study, we examined the effects of extracellular ATP on VR1. In cells expressing VR1, ATP increased the currents evoked by capsaicin or protons through activation of metabotropic P2Y1 receptors in a protein kinase C-dependent pathway. The involvement of Gq/11-coupled metabotropic receptors in the potentiation of VR1 response was confirmed in cells expressing both VR1 and M1 muscarinic acetylcholine receptors. In the presence of ATP, the temperature threshold for VR1 activation was reduced from 42°C to 35°C, such that normally nonpainful thermal stimuli (i.e., normal body temperature) were capable of activating VR1. This represents a novel mechanism through which the large amounts of ATP released from damaged cells in response to tissue trauma might trigger the sensation of pain.

Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-d-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERα) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERα and ERβ mRNA. At the light microscopic level, autoradiography shows cell nuclear [3H]estrogen and [125I]estrogen uptake according to a distribution that primarily reflects the localization of ERα-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERα immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.

Dopamine receptor regulating factor, DRRF: A zinc finger transcription factor

Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in brain with a specific regional distribution including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala, and frontal cortex. Many of these brain regions also express abundant levels of various dopamine receptors. In vivo, DRRF itself can be regulated by manipulations of dopaminergic transmission. Mice treated with drugs that increase extracellular striatal dopamine levels (cocaine), block dopamine receptors (haloperidol), or destroy dopamine terminals (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) show significant alterations in DRRF mRNA. The latter observations provide a basis for dopamine receptor regulation after these manipulations. We conclude that DRRF is important for modulating dopaminergic transmission in the brain.

GTP-binding protein βγ subunits mediate presynaptic calcium current inhibition by GABAB receptor

A variety of GTP-binding protein (G protein)-coupled receptors are expressed at the nerve terminals of central synapses and play modulatory roles in transmitter release. At the calyx of Held, a rat auditory brainstem synapse, activation of presynaptic γ-aminobutyric acid type B receptors (GABAB receptors) or metabotropic glutamate receptors inhibits presynaptic P/Q-type Ca2+ channel currents via activation of G proteins, thereby attenuating transmitter release. To identify the heterotrimeric G protein subunits involved in this presynaptic inhibition, we loaded G protein βγ subunits (Gβγ) directly into the calyceal nerve terminal through whole-cell patch pipettes. Gβγ slowed the activation of presynaptic Ca2+ currents (IpCa) and attenuated its amplitude in a manner similar to the externally applied baclofen, a GABAB receptor agonist. The effects of both Gβγ and baclofen were relieved after strong depolarization of the nerve terminal. In addition, Gβγ partially occluded the inhibitory effect of baclofen on IpCa. In contrast, guanosine 5′-O-(3-thiotriphosphate)-bound Goα loaded into the calyx had no effect. Immunocytochemical examination revealed that the subtype of G proteins Go, but not the Gi, subtype, is expressed in the calyceal nerve terminal. These results suggest that presynaptic inhibition mediated by G protein-coupled receptors occurs primarily by means of the direct interaction of Go βγ subunits with presynaptic Ca2+ channels.

Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.

The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation.

Norepinephrine transporters have channel modes of conduction.

Neurotransmitter transporters couple to existing ion gradients to achieve reuptake of transmitter into presynaptic terminals. For coupled cotransport, substrates and ions cross the membrane in fixed stoichiometry. This is in contrast to ion channels, which carry an arbitrary number of ions depending on the channel open time. Members of the gamma-aminobutyric acid transporter gene family presumably function with fixed stoichiometry in which a set number of ions cotransport with one transmitter molecule. Here we report channel-like events from a presumably fixed stoichiometry [norepinephrine (NE)+, Na+, and Cl-], human NE (hNET) in the gamma-aminobutyric acid transporter gene family. These events are stimulated by NE and by guanethidine, an hNET substrate, and they are blocked by cocaine and the antidepressant desipramine. Voltage-clamp data combined with NE uptake data from these same cells indicate that hNETs have two functional modes of conduction: a classical transporter mode (T-mode) and a novel channel mode (C-mode). Both T-mode and C-mode are gated by the same substrates and antagonized by the same blockers. T-mode is putatively electrogenic because the transmitter and cotransported ions sum to one net charge. However, C-mode carries virtually all of the transmitter-induced current, even though it occurs with low probability. This is because each C-mode opening transports hundreds of charges per event. The existence of a channel mode of conduction in a previously established fixed-stoichiometry transporter suggests the appearance of an aqueous pore through the transporter protein during the transport cycle and may have significance for transporter regulation.

Induction of long-term potentiation is associated with major ultrastructural changes of activated synapses.

Long-term potentiation (LTP), an increase in synaptic efficacy believed to underlie learning and memory mechanisms, has been proposed to involve structural modifications of synapses. Precise identification of the morphological changes associated with LTP has however been hindered by the difficulty in distinguishing potentiated or activated from nonstimulated synapses. Here we used a cytochemical method that allowed detection in CA1 hippocampus at the electron microscopy level of a stimulation-specific, D-AP5-sensitive accumulation of calcium in postsynaptic spines and presynaptic terminals following application of high-frequency trains. Morphometric analyses carried out 30-40 min after LTP induction revealed dramatic ultrastructural differences between labeled and nonlabeled synapses. The majority of labeled synapses (60%) exhibited perforated postsynaptic densities, whereas this proportion was only 20% in nonlabeled synaptic contacts. Labeled synaptic profiles were also characterized by a larger apposition zone between pre- and postsynaptic structures, longer postsynaptic densities, and enlarged spine profiles. These results add strong support to the idea that ultrastructural modifications and specifically an increase in perforated synapses are associated with LTP induction in field CA1 of hippocampus and they suggest that a majority of activated contacts may exhibit such changes.

Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry.

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.

Localization of alpha type II calcium calmodulin-dependent protein kinase at glutamatergic but not gamma-aminobutyric acid (GABAergic) synapses in thalamus and cerebral cortex.

The alpha subunit of type II calcium/calmodulin-dependent protein kinase (CAM II kinase-alpha) plays an important role in longterm synaptic plasticity. We applied preembedding immunocytochemistry (for CAM II kinase-alpha) and postembedding immunogold labeling [for glutamate or gamma-aminobutyric acid (GABA)] to explore the subcellular relationships between transmitter-defined axon terminals and the kinase at excitatory and inhibitory synapses in thalamus and cerebral cortex. Many (but not all) axon terminals ending in asymmetric synapses contained presynaptic CAM II kinase-alpha immunoreactivity; GABAergic terminals ending in symmetric synapses did not. Postsynaptically, CAM II kinase-alpha immunoreactivity was associated with postsynaptic densities of many (but not all) glutamatergic axon terminals ending on excitatory neurons. CAM II kinase-alpha immunoreactivity was absent at postsynaptic densities of all GABAergic synapses. The findings show that CAM II kinase-alpha is selectively expressed in subpopulations of excitatory neurons and, to our knowledge, demonstrate for the first time that it is only associated with glutamatergic terminals pre- and postsynaptically. CAM II kinase-alpha is unlikely to play a role in plasticity at GABAergic synapses.

Isoform-specific interaction of the alpha1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25.

Presynaptic Ca2+ channels are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. Here we report isoform-specific, stoichiometric interaction of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels with the presynaptic membrane proteins syntaxin and SNAP-25 in vitro and in rat brain membranes. The BI isoform binds to both proteins, while only interaction with SNAP-25 can be detected in vitro for the rbA isoform. The synaptic protein interaction ("synprint") site involves two adjacent segments of the intracellular loop connecting domains II and III between amino acid residues 722 and 1036 of the BI sequence. This interaction is competitively blocked by the corresponding region of the N-type Ca2+ channel, indicating that these two channels bind to overlapping regions of syntaxin and SNAP-25. Our results provide a molecular basis for a physical link between Ca2+ influx into nerve terminals and subsequent exocytosis of neurotransmitters at synapses that have presynaptic Ca2+ channels containing alpha1A subunits.