Kinetics of etomidate actions on GABA(A) receptors in the rat spinal dorsal horn neurons

Electrophysiological properties of etomidate (ET)-induced current (I(ET)) at different concentrations and effects of ET at clinically relevant concentrations (1-10 microM) on postsynaptic GABA(A) receptor function were investigated using whole-cell patch-clamp technique in mechanically dissociated rat spinal dorsal horn neurons. The results showed that ET actions were concentration-dependent: low concentrations (10 microM) of ET potentiated GABA-activated current (I(GABA)), slowed activation, desensitization and deactivation of GABA(A) receptors; moderate concentrations (10-1,000 microM) of ET directly activated and desensitized GABA(A) receptors; high concentrations (>1,000 microM) of ET produced an inhibitory effect on I(ET). In addition, ET prolonged the duration of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the mechanically dissociated rat dorsal horn neurons. These results suggest that general anesthetics-induced changes at spinal level could significantly contribute to analgesia and general anesthesia.     

Nonsegmental inhibition of rat dorsal horn neurons by innocuous stimulation

In rats anesthetized with thiamylal sodium, responses of spinal cord dorsal horn neurons to noxious skin heating of the tail were recorded by extracellular microelectrodes. Inhibition of these responses by innocuous mechanical stimulation (light brushing) of the ipsilateral forelimb was assessed. Short-lasting application (3 min, or less) of light brushing did not inhibit neuronal responses to noxious heating. Long-lasting application (5 min, or more) inhibited responses of these neurons to noxious stimulation. The results indicate that, in the anesthetized rat, remotely applied innocuous cutaneous stimuli can inhibit nociceptive responses of dorsal horn neurons, if applied for a sufficiently long time.     

Intrinsic firing properties of developing rat superficial dorsal horn neurons

Neonatal superficial dorsal horn neurons exhibit distinct firing properties in response to nociceptive and tactile inputs, but it is not known whether the intrinsic membrane excitability of these neurons changes during the early postnatal period. We have investigated the evoked firing properties of dorsal horn cells in rat spinal cord slices at different postnatal ages (P3, P10 and P21) and found no significant differences in mean firing frequency, spike frequency adaptation, regularity of action potential discharge or rheobase current levels between age groups. These results demonstrate that the intrinsic excitability of superficial dorsal horn neurons remains stable during early postnatal development and suggest that alterations in the synaptic inputs to these cells explain the changes in response to peripheral stimulation.     

Connectivity of pacemaker neurons in the neonatal rat superficial dorsal horn

Pacemaker neurons with an intrinsic ability to generate rhythmic burst‐firing have been characterized in lamina I of the neonatal spinal cord, where they are innervated by high‐threshold sensory afferents. However, little is known about the output of these pacemakers, as the neuronal populations that are targeted by pacemaker axons have yet to be identified. The present study combines patch‐clamp recordings in the intact neonatal rat spinal cord with tract‐tracing to demonstrate that lamina I pacemaker neurons contact multiple spinal motor pathways during early life. Retrograde labeling of premotor interneurons with the trans‐synaptic pseudorabies virus PRV‐152 revealed the presence of burst‐firing in PRV‐infected lamina I neurons, thereby confirming that pacemakers are synaptically coupled to motor networks in the spinal ventral horn. Notably, two classes of pacemakers could be distinguished in lamina I based on cell size and the pattern of their axonal projections. Whereas small pacemaker neurons possessed ramified axons that contacted ipsilateral motor circuits, large pacemaker neurons had unbranched axons that crossed the midline and ascended rostrally in the contralateral white matter. Recordings from identified spino‐parabrachial and spino‐periaqueductal gray neurons indicated the presence of pacemaker activity within neonatal lamina I projection neurons. Overall, these results show that lamina I pacemakers are positioned to regulate both the level of activity in developing motor circuits and the ascending flow of nociceptive information to the brain, thus highlighting a potential role for pacemaker activity in the maturation of pain and sensorimotor networks in the central nervous system. J. Comp. Neurol. 523:1038–1053, 2015. © 2015 Wiley Periodicals, Inc.     

Actions of serotonin recorded intracellularly in rat dorsal lateral septal neurons

The actions of serotonin (5HT) on passive and active membrane properties of neurons in the rat dorsal lateral septal nucleus (LSN) were studied by using intracellular recordings in transverse, septal slices. Superfusion with 10 microM 5HT induced a hyperpolarization of the membrane in almost all neurons tested in the dorsolateral part of the LSN. The hyperpolarization was accompanied by a decrease in membrane resistance. These effects of 5HT persisted in a low-Ca2+/high-Mg2+-containing medium or medium with tetrodotoxin, indicating a post-synaptic site of action for 5HT. The reversal potential for the hyperpolarizing effect was ca. -95 mV. If the extracellular K+-concentration was raised, the reversal potential became less negative. These data suggest that 5HT hyperpolarizes LSN neurons by increasing a K+-conductance. Spontaneous, synaptically evoked action potentials and action potentials induced in LSN neurons by a depolarizing current step typically display a fast Na+-spike with a subsequent K+-afterhyperpolarization, followed by a much slower Ca2+-dependent afterdepolarization. The amplitude of the K+-afterhyperpolarization was decreased by 5HT, while at the same time the afterdepolarization became more pronounced. The Ca2+-spike of LSN neurons was not affected by 5HT. Synaptic responses that were evoked in LSN neurons by stimulation of the dorsal part of the LSN consisted of a fast EPSP or spike, followed by a Cl(-)-dependent fast IPSP and a K+-dependent late IPSP. Of these synaptic responses, 5HT suppressed particularly the late IPSP. The present data indicate that 5HT affects the conductance for active and passive K+-channels in LSN neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recording of long-term potentiation in single dorsal horn neurons in vivo in the rat.

We have published several reports on long-term potentiation (LTP) in single spinal wide dynamic range (WDR) neurons (responding to both innocuous and noxious stimuli) in urethane-anaesthetised rats. The protocol presented here, with single unit recordings of dorsal horn neurons before and after a nociceptive conditioning stimulation, may be useful in many electrophysiological studies of plastic changes in the spinal cord, such as LTP. We invite others to use this protocol for the study of spinal plasticity. Findings using this technique may be relevant for the understanding of changes in nociceptive transmission, induction of central sensitisation and maybe even in mechanisms of pathological pain and chronic pain states. We describe modified and alternative protocols for the study of LTP mechanisms under different conditions in intact and in spinalised animals, and after natural noxious stimuli. We present a novel method minimising peripheral influence of afferent input induced by antidromic neurogenic inflammation or inflammatory changes following a natural noxious stimulation. This is made possible by dissection of the sciatic nerve at two separate locations and local anaesthetic block distal to the stimulation site.     

Role of cervical neurons in propriospinal inhibition of thoracic dorsal horn neurons.

We previously reported that electrical or glutamate stimulation of the cervical spinal cord elicits a 40-60% decrease in renal sympathetic nerve activity (RSA) in the anesthetized rats. This sympatho-inhibition was possible, however, only after transection of the spinal cord at C1 or GABAergic inhibition of neurons in the rostral ventrolateral medulla. We postulated that cervical neurons inhibit RSA by inhibiting the activity of spinal interneurons that are antecedent to sympathetic preganglionic neurons (SPNs), and that these interneurons may be, in turn, excited by afferent signals. In this study, we tested the hypothesis that cervical neurons can inhibit visceroceptive thoracic spinal neurons. We recorded the spontaneous and evoked activity of 45 dorsal horn neurons responsive to splanchnic stimulation before, during, and after chemical or electrical stimulation of the cervical spinal cord in chloralose-anesthetized spinal rats. Cervical spinal stimulation that inhibited RSA also inhibited the spontaneous and/or evoked activity of 44 dorsal horn neurons. In addition to inhibiting splanchnic-evoked neuronal responses, cervical stimulation also inhibited responses, in the same neurons, evoked by noxious heat or light brushing of receptive dermatomes. We concluded that cervical neurons participate in propriospinal inhibition of afferent transmission and that this inhibitory system may be involved in controlling the access of afferent information to SPNs.     

Modulation of visceral nociceptive responses of rat spinal dorsal horn neurons by sympathectomy

We determined whether sympathectomy modulates visceral nociception under physiological or inflammatory conditions. Recordings of sacral spinal dorsal horn neurons with sustained responses were performed in pentobarbitone-anesthetized rats. Graded colorectal distension (CRD, 20-100 mmHg) was used as a visceral nociceptive stimulus. Inflammation was induced by intracolonic instillation of turpentine (25%). Sympathectomy was produced by administering 6-hydroxydopamine. Inflammation produced an increase in the CRD-evoked responses. The CRD-evoked responses were attenuated following sympathectomy both under control and inflammatory conditions. These changes in the CRD-evoked responses were associated with corresponding changes in spontaneous discharge rate. The convergent input evoked by noxious pinch of the skin was not changed by any of the experimental conditions. The results indicate that sympathectomy attenuates visceral nociceptive responses and spontaneous activity of sacral spinal cord neurons, without effect on convergent cutaneous inputs, both under physiological and inflammatory conditions.     

Quantitative study of NPY-expressing GABAergic neurons and axons in rat spinal dorsal horn

Between 25-40% of neurons in laminae I-III are GABAergic, and some of these express neuropeptide Y (NPY). We previously reported that NPY-immunoreactive axons form numerous synapses on lamina III projection neurons that possess the neurokinin 1 receptor (NK1r). The aims of this study were to determine the proportion of neurons and GABAergic boutons in this region that contain NPY, and to look for evidence that they selectively innervate different neuronal populations. We found that 4-6% of neurons in laminae I-III were NPY-immunoreactive and based on the proportions of neurons that are GABAergic, we estimate that NPY is expressed by 18% of inhibitory interneurons in laminae I-II and 9% of those in lamina III. GABAergic boutons were identified by the presence of the vesicular GABA transporter (VGAT) and NPY was found in 13-15% of VGAT-immunoreactive boutons in laminae I-II, and 5% of those in lamina III. For both the lamina III NK1r-immunoreactive projection neurons and protein kinase Cγ (PKCγ)-immunoreactive interneurons in lamina II, we found that around one-third of the VGAT boutons that contacted them were NPY-immunoreactive. However, based on differences in the sizes of these boutons and the strength of their NPY-immunoreactivity, we conclude that these originate from different populations of interneurons. Only 6% of VGAT boutons presynaptic to large lamina I projection neurons that lacked NK1rs contained NPY. These results show that NPY-containing neurons make up a considerable proportion of the inhibitory interneurons in laminae I-III, and that their axons preferentially target certain classes of dorsal horn neuron.     

Neonatal rat spinal cord slice preparation: postsynaptic effects of neuropeptides on dorsal horn neurons

In the neonatal rat spinal cord slice preparation responses of the dorsal horn interneurons to iontophoretic or bath application of methionine-enkephalin (ME), substance P (SP) and somatostatin (SS) were qualitatively similar to those obtained in intact spinal cord. Thus, SP powerfully excited almost all neurons tested (15/16), while ME and SS depressed neuronal discharges in 13/14 and 4/6 units respectively. In some dorsal horn neurons the iontophoretic application of ME caused a marked depression of the SP-induced excitation. Angiotensin II (AgII) had no effect on dorsal horn units (n = 8). In the slices perfused with a Ca2+-free, Mg2+-high Krebs solution the extracellularly recorded effects of ME, SP and SS were not significantly modified, suggesting that the peptides were acting directly on postsynaptic sites. The results also indicate that the in vitro rat spinal cord slice preparation can be successfully utilized for further studies on the cellular mechanisms of actions of neuropeptides, particularly in relation to synaptic transmission processes in the dorsal horn.     

R(-)-baclofen: focal epilepsy after intracortical administration in the rat

R(-)- or S(+)-baclofen were injected into lamina IV-V of the sensorimotor cortex of the rat. Clinical observation and ECoG registration revealed that partial epilepsy with focal motor symptoms developed following injection of R(-)-baclofen, with an ED50 of 0.25 nmoles, a mean latency of 17 min independent of the dose, and a duration of more than 5 h at a dose of 5 nmoles. S(+)-Baclofen was ineffective at doses of up to 5 nmoles (2 x ED100 (-)-baclofen), indicating a stereoselective action of the (-)-isomer.     

Sodium salicylate reduces gamma aminobutyric acid-induced current in rat spinal dorsal horn neurons

Sodium salicylate is one of the nonsteroidal antiinflammatory drugs and is clinically used for antiinflammation and chronic pain relief. In the present study, we investigated the actions of sodium salicylate on gamma-aminobutyric acid type A receptor (GABA(A)) current in cultured rat spinal dorsal horn neurons. Sodium salicylate was found to reduce GABA(A) current in a reversible and concentration-dependent manner, but did not change its ion selectivity. Sodium salicylate was effective only when GABA and sodium salicylate were applied together. Application of sodium salicylate immediately before, but not during, the application of GABA did not result in a significant reduction of GABA(A) current. Our results demonstrate that sodium salicylate reversibly attenuates the GABA(A) response of dorsal horn neurons, suggesting that GABA(A) receptors in the region are pharmacological targets of sodium salicylate.     

Activation of calcium channel currents in rat sensory neurons by large depolarizations: effect of Guanine nucleotides and (-)-baclofen

Calcium channel currents have been recorded from cultured rat sensory neurons at clamp potentials of between -30 and +120 mV. At large depolarizing potentials between +50 and +120 mV, the current was outward. This outward current was shown to be largely due to ions passing through calcium channels, because it was substantially although generally incompletely blocked by Cd2+ (1 mM) and omega-conotoxin (1 microM). Internal GTP-gamma-S (100 microM) and to a lesser extent GTP (1 mM) reduced the amplitude and slowed the activation of the outward, as well as the inward calcium channel current. Baclofen (100 microM) reversibly inhibited both the inward and outward currents. These results suggest that the effect of baclofen and G protein activation on calcium channel currents is not due to a shift in the voltage-dependence of channel availability.     

Ascending projections from marginal zone (Lamina I) neurons of the spinal dorsal horn

A search for postsynaptic elements, excited only by slowly-conducting afferent fibers (Aδ and C), was made by recording from the dorsal horn of cats and monkeys with dye-filled microelectrodes. Units identified by afferent responsiveness were tested for antidromic invasion by electrical stimulation of the opposite ventrolateral funiculus at the midcervical level. Recording points were marked by iontophoretically passing dye from the recording electrode and subsequently located in histologically-prepared material. Evidence for antidromic excitation from spinal cord stimulation was found for five of 21 units in cat and 13 of 31 neurons in monkey. Antidromically excited cells were located within the distribution of the large posteromarginal neurons of the dorsal marginal zone (Lamina I). Although recording loci for a number of the elements studied were unequivocally located within Laminae II (substantia gelatinosa) or III, none could be antidromically excited. All antidromically driven units received a powerful input from Aδ primary afferent units and upon testing with natural stimuli responded specifically to stimuli of the type initiating activity either for high threshold mechanoreceptors or for low threshold thermoreceptors. It is concluded that some Lamina I neurons form part of an ascending projection which follows the spinal pathway of the spinothalamic tract and thereby contribute to the mechanical nociceptive and thermoreceptive features of this pathway. The absence of antidromic response is argued to be uncertain and evidence for a lack of projection is open to alternative explanation.     

Selective inhibition by systemic lidocaine of noxious evoked activity in rat dorsal horn neurons

The effect of systemically injected lidocaine (3-4 mg kg-1) on the responses to noxious and non-noxious stimuli on 28 wide dynamic range (WDR) neurons in the dorsal horn was studied in anesthetized and curarized rats. It was consistently found that lidocaine reduced or suppressed the responses to noxious stimuli whereas it did not act on the responses to non-noxious stimulation and on the spontaneous activity. Furthermore the noxious stimuli were completely ineffective from 10-15 min following the lidocaine injection while the non-noxious stimuli maintained their efficacy. The control responses, in all the cases, returned within 20 min. The results suggest that lidocaine exerts a selective inhibitory effect on nociceptive transmission at the spinal level.     

Inhibition of rat spinal cord dorsal horn neurons by non-segmental, noxious cutaneous stimuli

Extracellular single unit recordings were obtained from spinal cord dorsal horn neurons in halothane-anesthetized rats. Inhibitory effects induced by noxious mechanical or electrical stimuli applied to a remote area of the body surface were assessed on the spontaneous or evoked activity of these cells. Noxious mechanical stimulation inhibited 59% of the cells receiving nociceptive inputs (wide dynamic range and nociceptive specific) but only 5% of the other cell types. Inhibition produced by mechanical stimulation lasted for the full duration of stimulus application (up to 30 s) whereas inhibition produced by electrical stimulation lasted less than 500 ms. Increasing the depth of anesthesia was found to depress or abolish the inhibition.     

Subpopulations of GABAergic and non-GABAergic rat dorsal horn neurons express Ca2+-permeable AMPA receptors.

Subpopulations of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors that are either permeable or impermeable to Ca2+ are expressed on dorsal horn neurons in culture. While both mediate synaptic transmission, the Ca2+ -permeable AMPA receptors provide a Ca2+ signal that may result in a transient change in synaptic strength [Gu, J.G., Albuquerque, C., Lee, C.J. & MacDermott, A.B. (1996) Nature, 381, 793]. To appreciate the relevance of these receptors to dorsal horn physiology, we have investigated whether they show selective expression in identified subpopulations of dorsal horn neurons. Expression of Ca2+-permeable AMPA receptors was assayed using the kainate-induced cobalt loading technique first developed by Pruss et al. [Pruss, R.M., Akeson, R.L., Racke, M.M. & Wilburn, J.L. (1991) Neuron, 7, 509]. Subpopulations of dorsal horn neurons were identified using immunocytochemistry for gamma-aminobutyric acid (GABA), glycine, substance P receptor (NK1 receptor) and the Ca2+-binding proteins, calretinin and calbindin D28K. We demonstrate that, in dorsal horn neurons in culture, kainate-induced cobalt uptake is selectively mediated by Ca2+-permeable AMPA receptors, and that a majority of GABA and NK1 receptor-expressing neurons express Ca2+-permeable AMPA receptors. GABAergic dorsal horn neurons are important in local inhibition as well as in the regulation of transmitter release from primary afferent terminals. NK1 receptor-expressing dorsal horn neurons include many of the projection neurons in the nociceptive spino-thalamic pathway. Thus, we have identified two populations of dorsal horn neurons representing important components of dorsal horn function that express Ca2+-permeable AMPA receptors. Furthermore, we show that several subpopulations of putative excitatory interneurons defined by calretinin and calbindin expression do not express Ca2+-permeable AMPA receptors.     

Membrane properties of deep dorsal horn neurons from neonatal rat spinal cord in vitro

Whole-cell patch-clamp recordings were undertaken to characterize and compare the membrane properties of deep dorsal horn neurons in transverse slices of rat lumbar spinal cord in two age groups, postnatal days (P) 3–6 and 9–16. In both age groups, significant correlations were observed between membrane time constant and cell resistance and between action potential height and its duration at half-maximal amplitude. Cell resistance and action potential half-width values were lower in the P9–16 age group. Neurons were divided into four categories based on their firing properties in response to intracellular current injection: single spike, phasic firing, repetitive firing, and delayed firing. The distribution of neurons within these categories was similar in both age groups which suggests that the firing properties of deep dorsal horn neurons are functionally differentiated at an early postnatal age.     

Responses of rat dorsal horn neurons to natural stimulation and to iontophoretically applied norepinephrine

Extracellular recordings were obtained of 177 neurons throughout the lumbar spinal dorsal horn of urethane- or halothane-anesthetized rats. These neurons all responded to iontophoretically applied L-glutamate and their responses to natural stimulation of the ipsilateral hindlimb were characterized. Iontophoretically applied norepinephrine was tested on 94 of these neurons. Fifty-one neurons were inhibited and 22 were excited. Norepinephrine produced a biphasic inhibitory/excitatory effect on nine neurons. Norepinephrine was exclusively inhibitory on superficial dorsal horn neurons that responded only to innocuous brush and touch and on neurons in the nucleus proprius that responded to brush, touch, and noxious skin pinch. Norepinephrine excited some superficial brush/touch/pinch neurons and produced short inhibitions that were followed by prolonged excitations of some nucleus proprius neurons that responded only to noxious skin pinch. Neurons in the base of the dorsal horn that responded to low-threshold proprioceptive stimulation were excited by norepinephrine. Both the inhibitory and excitatory effects of norepinephrine were stereoselective, but they were not blocked by receptor subtype-selective antagonists. Desensitization to norepinephrine occurred for 30% of the neurons. This study demonstrates that the inhibitory effects of norepinephrine on rat dorsal horn neurons are not restricted to neurons that are responsive to noxious stimuli and that some of these neurons are primarily excited by norepinephrine. The excitatory effects of norepinephrine on low-threshold proprioceptive neurons may contribute to norepinephrine's known enhancement of spinal flexor reflex activity.     

Extension of dorsal horn neurons into the severed and implanted dorsal root

After dorsal root ganglionectomy in adult rats, the dorsal root was cut close to the spinal cord and implanted into the dorsal horn. Outgrowth from neurons in he dorsal horn of the spinal cord into the implanted dorsal root could be demonstrated after 3 months by means of retrograde HRP labeling. Double-labeling experiments showed that some of these neurons had retained their central projections while extending new processes into the implanted root. The possibility to reconstruct the sensory pathway by replacing the damaged primary sensory neuron with peripheral outgrowth from secondary sensory neurons is discussed.