GlyT-2 mediates the forskolin-induced increase of glycinergic transmission.

In this study, we have examined the possible roles of a glycine transporter type 2 (GlyT-2) in the forskolin-induced increase of the amplitude of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in acutely isolated rat substantia gelatinosa neurons. Forskolin, an adenylyl cyclase activator, increased the amplitude of glycinergic mIPSCs in the presence, but not in the absence, of a low concentration of extracellular glycine. This effect disappeared by the addition of ALX1393 (a GlyT-2 antagonist). These results suggest that both extracellular glycine and GlyT-2 are essential for the forskolin-induced increase in the amplitude of glycinergic mIPSCs. These mechanisms might contribute, at least in part, to the maturation of inhibitory synaptic transmission, including the developmental neurotransmitter switch from GABA to glycine within the spinal dorsal horn during postnatal development.

Cyclic AMP-mediated long-term facilitation of glycinergic transmission in developing spinal dorsal horn neurons.

cAMP is known to regulate neurotransmitter release via protein kinase A (PKA)-dependent and/or PKA-independent signal transduction pathways at a variety of central synapses. Here we report the cAMP-mediated long-lasting enhancement of glycinergic transmission in developing rat spinal substantia gelatinosa neurons. Forskolin, an adenylyl cyclase activator, elicited a long-lasting increase in the amplitude of nerve-evoked glycinergic inhibitory postsynaptic currents (IPSCs), accompanied by a long-lasting decrease in the paired-pulse ratio in immature substantia gelatinosa neurons, and this forskolin-induced increase in glycinergic IPSCs decreased with postnatal development. Forskolin also decreased the failure rate of glycinergic IPSCs evoked by minimal stimulation, and increased the frequency of glycinergic miniature IPSCs. All of these data suggest that forskolin induces the long-lasting enhancement of glycinergic transmission by increasing in the presynaptic release probability. This pre-synaptic action of forskolin was mediated by hyperpolarization and cyclic nucleotide-activated cation channels and an increase in intraterminal Ca(2+) concentration but independent of PKA. The present results suggest that cAMP-dependent signal transduction pathways represent a dynamic mechanism by which glycinergic IPSCs could potentially be modulated during postnatal development.

Alterations in primary afferent input to substantia gelatinosa of adult rat spinal cord after neonatal capsaicin treatment.

Primary afferent fibers are divided into three main subgroups: Abeta-, Adelta-, and C-fibers. Morphological studies have demonstrated that neonatal capsaicin treatment (NCT) depletes C-fiber inputs in the spinal dorsal horn; the electrophysiological features of the NCT-induced changes, however, remain unclear. This issue was addressed by performing whole-cell voltage-clamp recordings from substantia gelatinosa (SG) neurons in dorsal root-attached spinal cord slices. When estimated from excitatory postsynaptic currents (EPSCs) evoked by stimulating primary afferent fibers, 24 (49%) of 49 neurons examined exhibited C-fiber EPSCs that were either monosynaptic (n = 15) or polysynaptic (n = 9) in origin; only two of the neurons had Abeta-fiber responses. In NCT rats, however, SG neurons exhibiting C-fiber-mediated EPSCs decreased to 7% (3 of 41 neurons tested), whereas Abeta-fiber EPSCs were observed in 21 (51%) of the neurons, and 14 (67%) of them exhibited monosynaptic ones. There was no change in the cell proportion having Adelta-fiber innervation after NCT. Our electrophysiological data suggest that NCT diminishes primary afferent C-fiber inputs while enhancing Abeta-fiber direct innervation in the SG in adulthood.

Developmental changes in P2X purinoceptors on glycinergic presynaptic nerve terminals projecting to rat substantia gelatinosa neurones.

1. In mechanically dissociated rat spinal cord substantia gelatinosa (SG) neurones attached with native presynaptic nerve endings, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using nystatin perforated patch recording mode under voltage-clamp conditions. Under these conditions, it was tested whether the changes in P2X receptor subtype on the glycinergic presynaptic nerve terminals occur during postnatal development. 2. ATP facilitated glycinergic mIPSC frequency in a concentration-dependent manner through all developmental stages tested, whereas alphabeta-methylene-ATP (alphabeta-me-ATP) was only effective at later developmental stages. 3. alphabeta-me-ATP-elicited mIPSC frequency facilitation was completely occluded in the Ca2+-free external solution, but it was not affected by adding 10(-4) M Cd2+. 4. alphabeta-me-ATP still facilitated mIPSC frequency even in the presence of 10(-6) M thapsigargin, a Ca2+ pump blocker. 5. In later developmental stages, ATP-elicited presynaptic or postsynaptic responses were reversibly blocked by 10(-5) M pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), but only partially blocked by 10(-7) M 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP). However, alphabeta-me-ATP-elicited presynaptic or postsynaptic responses were completely and reversibly blocked by either 10(-5) M PPADS or 10(-7) M TNP-ATP. 6. alphabeta-me-ATP significantly reduced the evoked glycinergic IPSC amplitude in postnatal 28-30 day neurones, whereas it had no effect in 10-12 day neurones. 7. It was concluded that alphabeta-me-ATP-sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals projecting to SG neurones in later developmental stages. Such developmental changes of presynaptic P2X receptor subtypes might contribute to synaptic plasticity such as the regulation of neuronal excitability and the fine controlling of the pain signal in spinal dorsal horn neurones.

Alteration in synaptic inputs through C-afferent fibers to substantia gelatinosa neurons of the rat spinal dorsal horn during postnatal development.

The change in synaptic inputs through primary afferent C- and A-fibers during postnatal development was examined in substantia gelatinosa neurons of a rat spinal cord slice with an attached L5 dorsal root by use of the blind whole-cell patch-clamp technique; the synaptic responses were compared between the slices obtained from immature (postnatal days 21-23) and mature (postnatal days 56-60) male rats. The mono- and/or polysynaptic afferent inputs were monitored by recording glutamatergic excitatory postsynaptic currents and potentials evoked by stimulating C- and A-fibers, the identification of which was based on the values of threshold stimulus intensity and of the conduction velocity of the fibers, determined by intracellular recordings from dorsal root ganglion neurons. Immature substantia gelatinosa neurons received synaptic inputs through Abeta-, Adelta- and C-afferents, with proportions of 51%, 46% and 36%, respectively. In mature substantia gelatinosa neurons, C- and Adelta-afferent inputs were increased in number (to 84% and 86%, respectively), while Abeta-inputs were decreased to 9%. In both immature and mature rats, repetitive stimulation of C-afferents did not elicit any slow responses, which are longer in duration than the monosynaptic excitatory postsynaptic currents, although C-fibers are known to contain not only excitatory amino acids, but also neuropeptides such as substance P, which is thought to be involved in the production of slow responses. These results indicate that both C- and Adelta-afferents innervating substantia gelatinosa neurons are reorganized following maturation, accompanied by a withdrawal or elimination of Abeta-fibers from the substantia gelatinosa, probably due to a competition among the fibers during development. In spite of the developmental increase in C-fiber inputs, mature as well as immature substantia gelatinosa neurons did not display any slow synaptic responses, which appear to be mediated by transmitters other than excitatory amino acids.

Neonatal capsaicin treatment prevents the normal postnatal withdrawal of A fibres from lamina II without affecting fos responses to innocuous peripheral stimulation.

The development of spinal cord sensory pathways has been investigated in postnatal day (P) 21 rat pups following neonatal capsaicin treatment. Capsaicin-induced destruction of C fibres was confirmed by 62% loss of Isolectin B4 (IB4)-binding and an 86% loss of calcitonin gene-related peptide (CGRP)-immunoreactive small diameter dorsal root ganglion cells. Neonatal capsaicin treatment prevented the normal withdrawal of choleragenoid-horseradish peroxidase (B-HRP)-labelled A fibres from lamina II (substantia gelatinosa) to deeper laminae postnatally. A fibre terminals projected more dorsally, extending into 43% of lamina II compared to vehicle-treated littermates. A small cell loss in, and/or shrinkage of, substantia gelatinosa cannot account for this. These support the concept of a competitive interaction between A and C fibre afferents to establish final terminal fields. However the continued exuberant A fibre termination in capsaicin-treated rats did not lead to continued c-fos induction in the superficial dorsal horn by innocuous stimulation. In normal development, exuberant A fibre terminals coincide with c-fos activation in lamina II by innocuous skin stimulation [23]. Despite the continued presence of exuberant A fibre terminals, c-fos was not induced by innocuous peripheral stimulation in P21 capsaicin-treated rats implying that these superficial terminals do not activate lamina II neurons in the same way as in the neonate.

Reorganization of the primary afferent termination in the rat spinal dorsal horn during post-natal development.

To study the reorganization of the primary afferent input in the spinal dorsal horn during post-natal development, synaptic responses evoked by large Abeta and fine Adelta afferents were recorded from substantia gelatinosa (SG) neurons in slices obtained from immature (post-natal days 21-23) and mature rats (post-natal days 56-60). Threshold stimulus intensities and conduction velocities (CVs) of Abeta and Adelta afferents were determined by intracellular recordings of the antidromic action potentials from dorsal root ganglion (DRG) neurons isolated from immature and mature rats. In immature rats, excitatory postsynaptic currents (EPSCs) were elicited by stimulation sufficient to activate Abeta afferents in the majority of SG neurons (64.9%, 24 of 37 neurons), while most EPSCs observed in mature rats were elicited by stimulation of Adelta afferents (62.5%, 25 of 40 neurons). These observations suggest that the primary afferents innervating SG neurons were reorganized following maturation; Abeta afferents were the predominant inputs to the SG neurons in the immature state, thereafter Adelta afferents were substituted for the Abeta afferents to convey sensory information to the SG neurons. This relatively slow reorganization of the sensory circuitry may correlate with slow maturation of the SG neurons and with a delay in the functional connections of C afferents to the SG neurons.

Developmental alterations in NMDA receptor-mediated [Ca2+]i elevation in substantia gelatinosa neurons of neonatal rat spinal cord.

Using spinal cord slices prepared from neonatal rats, the intracellular free Ca2+ concentration ([Ca2+]i) in neurons located in the dorsal horn substantia gelatinosa (SG) was measured with microscopic fluorometry by loading fura 2-AM into neurons. Developmental alterations in the elevation of [Ca2+]i elicited by the glutamate analogs, NMDA and AMPA, were investigated from postnatal day (PNDs) 1 to 17. During the 1st week of postnatal life, when neuronal maturation of the SG is known to take place, the NMDA response remained large or even slightly increased. It subsequently showed a gradual decline. This pattern of postnatal changes is consistent with previously reported autoradiographic studies on NMDA-binding sites. The affinity of receptors for NMDA was found to decrease constantly during the period examined. The AMPA response and resting [Ca2+]i showed no significant developmental changes. Neonatal treatment with capsaicin, which has been shown to degenerate fine primary afferent fibers terminating in the SG, delayed the developmental decline in the NMDA-induced [Ca2+]i response. It is suggested that the number and the molecular properties of NMDA receptors expressed in the SG change during early postnatal neuronal maturation. The temporal coincidence between postnatal alteration in NMDA-induced [Ca2+]i elevation and neuronal maturation of the SG may indicate that intracellular Ca2+ regulated by NMDA receptor activation is related to postnatal neuronal maturation. Activation of fine primary afferent fibers may contribute to the observed developmental alterations in the NMDA response of SG neurons.

Neurons with asymmetrical dendritic arbors in the substantia gelatinosa of the rat spinal cord.

Two types of neurons were observed in the substantia gelatinosa (SG) of the rat spinal cord which exhibit wide variations in dendritic symmetry. As demonstrated with the Golgi technique, "islet" cells with short dendritic arbors and "type III stalk" cells display dendritic patterns which vary from a bipolar type arrangement with two dendritic arbors of nearly equal dimensions to a unipolar arrangement with a dendritic arbor which extends in only one direction. Examination of the morphology and dendritic development of these neurons shows that they are unique compared with other SG neurons in that they have short, longitudinal dendritic arbors which undergo maturation relatively late in the postnatal period. As is discussed, variations in dendritic symmetry are probably dependent on the location of the terminal fields of primary and/or other types of afferents which are formed earlier in development.

Postnatal maturation of primary afferent terminations in the substantia gelatinosa of the rat spinal cord. An electron microscopic study.

Axodendritic dark sinuous endings occurred on the day of birth (PO) in the synaptic areas of lamina II. These terminals (TI) turned very electron dense and shrunken after capsaicin administration. From day P2, TI-terminals exhibited fluoride-resistant acid phosphatase (FRAP) reactivity. Such findings revealed their origin from primary afferent fine fibers. Dark scalloped, FRAP-reactive, central terminals of type I glomeruli (CI) were first observed on P5. During the ensuing survival times up to P20 an increasing number of CI-terminals seemed to evolve from the less mature TI-endings that gradually disappeared. A few large clear boutons, similar to the central terminals of type II glomeruli (CII) of the adult which arise from thick afferents, were also present in deep lamina II on the day of birth and became more numerous thereafter. From P5, both CI- and CII-endings developed pre- and postsynaptic contacts with vesicle-containing profiles though it was sometimes difficult to distinguish the axonic (V2) from the dendritic (V1-presynaptic dendritic) profiles. CI-boutons established as many presynaptic as postsynaptic contacts with vesicle-containing profiles. In contrast, CII-terminals were mostly postsynaptic to vesicle-containing profiles. Thus, the boutons generated by thin (CI-boutons) and thick (CII-boutons) primary axons gradually develop synapses with vesicle-containing profiles probably arising from local interneurons. The resulting pre- and/or postsynaptic interactions may contribute to the physiological maturation of somatosensory integration that occurs postnatally.

Golgi and EM studies of the formation of dendritic and axonal arbors: the interneurons of the substantia gelatinosa of Rolando in newborn kittens.

Golgi studies in newborn kittens show that the two most prevalent interneurons in Rexed's lamina II of the dorsal horn of the medulla, the stalked cell and islet cell (Gobel, '75a,b, '78b) form their dendritic arbors in a similar fashion. At birth, both cell types are present in forms ranging from immature, in which numerous short dendrites radiate from the cell body in all directions, to relatively mature in which their dendritic arbors have elongated in specific directions and the adult branching pattern is already evident. During postnatal maturation, many dendrites are lost while only a few go on to lengthen. The unmyelinated axons of both cells are first recognized in forms in which lengthening dendrites have taken on their preferred direction of orientation. The two parts of Rexed's lamina II, i.e., layers IIa and IIb have already reached their adult mediolateral width at birth and the neuropil has nearly achieved its adult compactness. Space in the compact neuropil for elongating neuronal and astrocytic processes becomes available through the disintegration of many existing dendrites and by an overall fourfold increase in the rostrocaudal length of the dorsal horn of the medulla during postnatal maturation. At birth, the lengthening of the plasma membranes of elongating neuronal and astrocytic processes proceeds as vesicles (addition vesicles) found in aggregates throughout dendrites, unmyelinated axons and astrocytic processes fuse with and become incorporated into the existing plasma membranes. In addition, many dendrites in layers IIa and IIb are beading up and disintegrating. Within the beads, neurotubules are lost and addition vesicles fuse with each other to form small cavities. These cavities continue to enlarge, hollowing out the beads. The cavities ultimately open to the intercellular space as their membranes fuse with the plasma membrane of the beads. Finally, the beads disintegrate and their plasma membranes fragment. The thread-like segments between adjacent disintegrating beads shrivel until they ultimately disappear. Disintegration of beaded dendrites results in very little debris and does not provoke a phagocytic glial reaction. The disintegration of a dendritic branch takes place in spite of synaptic input from non-primary axons and is thought to occur from a failure to establish synaptic connections with primary axonal endings.