Polysynaptic Excitatory Postsynaptic Currents Research Articles

Increased Presynaptic and Postsynaptic α2-Adrenoceptor Activity in the Spinal Dorsal Horn in Painful Diabetic Neuropathy

Diabetic neuropathy is a common cause of chronic pain that is not adequately relieved by conventional analgesics. The α(2)-adrenoceptors are involved in the regulation of glutamatergic input and nociceptive transmission in the spinal dorsal horn, but their functional changes in diabetic neuropathy are not clear. The purpose of the present study was to determine the plasticity of presynaptic and postsynaptic α(2)-adrenoceptors in the control of spinal glutamatergic synaptic transmission in painful diabetic neuropathy. Whole-cell voltage-clamp recordings of lamina II neurons were performed in spinal cord slices from streptozotocin-induced diabetic rats. The amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked from the dorsal root and the frequency of spontaneous EPSCs (sEPSCs) were significantly higher in diabetic than vehicle-control rats. The specific α(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK-14304) (0.1-2 μM) inhibited the frequency of sEPSCs more in diabetic than vehicle-treated rats. UK-14304 also inhibited the amplitude of evoked monosynaptic and polysynaptic EPSCs more in diabetic than control rats. Furthermore, the amplitude of postsynaptic G protein-coupled inwardly rectifying K(+) channel (GIRK) currents elicited by UK-14304 was significantly larger in the diabetic group than in the control group. In addition, intrathecal administration of UK-14304 increased the nociceptive threshold more in diabetic than vehicle-control rats. Our findings suggest that diabetic neuropathy increases the activity of presynaptic and postsynaptic α(2)-adrenoceptors to attenuate glutamatergic transmission in the spinal dorsal horn, which accounts for the potentiated antinociceptive effect of α(2)-adrenoceptor activation in diabetic neuropathic pain.

Bone cancer induces a unique central sensitization through synaptic changes in a wide area of the spinal cord.

BackgroundChronic bone cancer pain is thought to be partly due to central sensitization. Although murine models of bone cancer pain revealed significant neurochemical changes in the spinal cord, it is not known whether this produces functional alterations in spinal sensory synaptic transmission. In this study, we examined excitatory synaptic responses evoked in substantia gelatinosa (SG, lamina II) neurons in spinal cord slices of adult mice bearing bone cancer, using whole-cell voltage-clamp recording techniques.ResultsMice at 14 to 21 days after sarcoma implantation into the femur exhibited hyperalgesia to mechanical stimuli applied to the skin of the ipsilateral hind paw, as well as showing spontaneous and movement evoked pain-related behaviors. SG neurons exhibited spontaneous excitatory postsynaptic currents (EPSCs). The amplitudes of spontaneous EPSCs were significantly larger in cancer-bearing than control mice without any changes in passive membrane properties of SG neurons. In the presence of TTX, the amplitude of miniature EPSCs in SG neurons was increased in cancer-bearing mice and this was observed for cells sampled across a wide range of lumbar segmental levels. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor- and N-methyl-D-aspartate (NMDA) receptor-mediated EPSCs evoked by focal stimulation were also enhanced in cancer-bearing mice. Dorsal root stimulation elicited mono- and/or polysynaptic EPSCs that were caused by the activation of Aδ and/or C afferent fibers in SG neurons from both groups of animals. The number of cells receiving monosynaptic inputs from Aδ and C fibers was not different between the two groups. However, the amplitude of the monosynaptic C fiber-evoked EPSCs and the number of SG neurons receiving polysynaptic inputs from Aδ and C fibers were increased in cancer-bearing mice.ConclusionsThese results show that spinal synaptic transmission mediated through Aδ and C fibers is enhanced in the SG across a wide area of lumbar levels following sarcoma implantation in the femur. This widespread spinal sensitization may be one of the underlying mechanisms for the development of chronic bone cancer pain.

Sustained Inhibition of Neurotransmitter Release from Nontransient Receptor Potential Vanilloid Type 1-Expressing Primary Afferents by μ-Opioid Receptor Activation-Enkephalin in the Spinal Cord

Removing transient receptor potential vanilloid type 1 (TRPV1)-expressing primary afferent neurons reduces presynaptic mu-opioid receptors but potentiates opioid analgesia. However, the sites and underlying cellular mechanisms for this paradoxical effect remain uncertain. In this study, we determined the presynaptic and postsynaptic effects of the mu-opioid receptor agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO) using whole-cell patch-clamp recordings of lamina II neurons in rat spinal cord slices. Treatment with the ultrapotent TRPV1 agonist resiniferotoxin (RTX) eliminated TRPV1-expressing dorsal root ganglion neurons and their central terminals in the spinal dorsal horn and significantly reduced the basal amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked from primary afferents. Although RTX treatment did not significantly alter the concentration-response effect of DAMGO on evoked monosynaptic and polysynaptic EPSCs, it causes a profound long-lasting inhibitory effect of DAMGO on evoked EPSCs. Subsequent naloxone treatment did not reverse the prolonged inhibitory effect of DAMGO on evoked EPSCs. Furthermore, brief application of DAMGO produced a sustained inhibition of miniature EPSCs in RTX-treated rats. However, the concentration response and the duration of the effects of DAMGO on G protein-coupled inwardly rectifying K+ currents in lamina II neurons were not significantly different between vehicle- and RTX-treated groups. These data suggest that stimulation of mu-opioid receptors on non-TRPV1 afferent terminals causes extended inhibition of neurotransmitter release to spinal dorsal horn neurons. The differential effect of mu-opioid receptor agonists on different phenotypes of primary afferents provides a cellular basis to explain why the analgesic action of opioids on mechanonociception is prolonged when TRPV1-expressing primary afferents are removed.

Optical and electrophysiological recordings of corticospinal synaptic activity and its developmental change in in vitro rat slice co-cultures

Electrophysiological recordings and optical imaging with a fast voltage-sensitive dye (di-4-ANNEPS) were used to directly examine the spatiotemporal properties of in vitro corticospinal synapses formed in co-cultures of cerebral cortex and spinal cord slices. Whole cell recordings from spinal cord cells showed both monosynaptic and polysynaptic excitatory postsynaptic currents (EPSCs) in response to stimulation of corticospinal axons. Monosynaptic EPSCs and excitatory postsynaptic potentials (EPSPs) were isolated in artificial cerebrospinal fluid containing high concentrations of divalent cations. Optical imaging and extracellular recordings were done simultaneously. Both EPSPs and optically recorded excitatory postsynaptic potentials (optEPSPs) lasted 300–500 ms and were almost always positive. The major component of these long-lasting potentials was blocked by ifenprodil, a specific antagonist of the NR2B subunit-containing N-methyl- d-aspartate receptor (NMDAR). The spatial distribution of corticospinal optEPSPs paralleled that of the corticospinal field excitatory postsynaptic potentials (fEPSPs), suggesting that positive fEPSP amplitude is a reliable indicator of the distribution of corticospinal synapses. Corticospinal optEPSPs spread into the ventrolateral region by 6–7 days in vitro (DIV), but were restricted to the dorsomedial area by 11–13 DIV, suggesting synapses were eliminated from the ventrolateral side of the spinal cord. After the recordings were complete, corticospinal fibers were often anterogradely labeled with biocytin to assess the relation between presynaptic fiber distribution and the optical signals (optically-recorded presynaptic fiber volley (opt-prevolley) and optEPSP). The distributions of the opt-prevolleys and optEPSPs correlated well with the distribution of presynaptic fibers, suggesting the opt-prevolley reflects corticospinal fiber activity and that the fibers made synapses relatively evenly along their axons. The NR2B-mediated component of the corticospinal synaptic response declined during the interval between 6 and 7 DIV and 11–13 DIV, suggesting that a shift in the NMDAR subtype from NR2B to something else (perhaps NR2A) may be involved in regulating developmental plasticity in the rat spinal cord and the process of corticospinal synapse elimination.

Actions of Midazolam on Excitatory Transmission in Dorsal Horn Neurons of Adult Rat Spinal Cord

Although intrathecal administration of midazolam, a water-soluble imidazobenzodiazepine derivative, has been found to produce analgesia, how it exerts this effect at the neuronal level in the spinal cord is not fully understood. The effects of midazolam on electrically evoked and spontaneous excitatory transmission were examined in lamina II neurons of adult rat spinal cord slices using the whole cell patch clamp technique. Bath-applied midazolam (1 microm) diminished Adelta- and C-fiber evoked polysynaptic excitatory postsynaptic currents in both amplitude and integrated area. However, it affected neither Adelta- and C-fiber evoked monosynaptic excitatory postsynaptic currents in amplitude nor miniature excitatory postsynaptic currents in amplitude, frequency, and decay time constant. In the presence of a benzodiazepine receptor antagonist, flumazenil (5 microm), midazolam (1 microm) did not diminish Adelta-fiber evoked polysynaptic excitatory postsynaptic currents, suggesting that midazolam modulate the gamma-aminobutyric acid interneurons in the dorsal horn. Midazolam reduced excitatory synaptic transmission by acting on the gamma-aminobutyric acid type A/benzodiazepine receptor in interneurons, leading to a decrease in the excitability of spinal dorsal horn neurons. This may be a possible mechanism for the antinociception by midazolam in the spinal cord.

Granule cell hyperexcitability in the early post-traumatic rat dentate gyrus: the 'irritable mossy cell' hypothesis.

1. Cytochemical and in vitro whole-cell patch clamp techniques were used to investigate granule cell hyperexcitability in the dentate gyrus 1 week after fluid percussion head trauma. 2. The percentage decrease in the number of hilar interneurones labelled with either GAD67 or parvalbumin mRNA probes following trauma was not different from the decrease in the total population of hilar cells, indicating no preferential survival of interneurones with respect to the non-GABAergic hilar cells, i.e. the mossy cells. 3. Dentate granule cells following trauma showed enhanced action potential discharges, and longer-lasting depolarizations, in response to perforant path stimulation, in the presence of the GABAA receptor antagonist bicuculline. 4. There was no post-traumatic alteration in the perforant path-evoked monosynaptic excitatory postsynaptic currents (EPSCs), or in the intrinsic properties of granule cells. However, after trauma, the monosynaptic EPSC was followed by late, polysynaptic EPSCs, which were not present in controls. 5. The late EPSCs in granule cells from fluid percussion-injured rats were not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), but were eliminated by both the non-NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the AMPA receptor antagonist GYKI 53655. 6. In addition, the late EPSCs were not present in low (0.5 mM) extracellular calcium, and they were also eliminated by the removal of the dentate hilus from the slice. 7. Mossy hilar cells in the traumatic dentate gyrus responded with significantly enhanced, prolonged trains of action potential discharges to perforant path stimulation. 8. These data indicate that surviving mossy cells play a crucial role in the hyperexcitable responses of the post-traumatic dentate gyrus.

Long-term potentiation of primary afferent neurotransmission at trigeminal synapses of juvenile rats.

Primary afferent monosynaptic and polysynaptic excitatory postsynaptic currents (EPSCs) were recorded from brainstem trigeminal neurons by stimulation of the mandibular nerve attached to the brainstem preparation of juvenile rats. A high-frequency conditioning stimulus induced long-term potentiation (LTP) of high-threshold EPSCs in the majority of trigeminal caudal neurons in substantia gelatinosa, where both A- and C-fibres terminate. However, the same conditioning stimulus did not potentiate low-threshold EPSCs in caudal neurons or EPSCs recorded from neurons in the middle part of trigeminal interpolar nucleus, where C-fibres rarely terminate. LTP in caudal neurons could be induced after blocking N-methyl-D-aspartate (NMDA) receptors with D(-)-2-amino-5-phosphonopentanoic acid (D-AP5, 50 microM), after postsynaptic loading of the Ca2+ chelator BAPTA (10 mM), or even after completely blocking excitatory transmission with kynurenic acid during conditioning. However, LTP was blocked by the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (1 mM). We suggest that LTP of the trigeminal primary afferent EPSCs is induced preferentially in the C-fibre inputs and that the induction mechanism involves metabotropic glutamate receptors, possibly at the presynaptic terminals.

Synaptic input from CA3 pyramidal cells to dentate basket cells in rat hippocampus.

1. Excitatory inputs from CA3 pyramidal cells to dentate basket cells were examined using the whole-cell recording technique in neonatal (10-16 days) rat hippocampal slices to characterize this unexpected feedback pathway. 2. Minimal electrical stimulation of the CA3 pyramidal layer evoked in basket cells short latency (5.2 +/- 0.4 ms) glutamate receptor-mediated excitatory postsynaptic currents (EPSCs) with fast rise times (at -70 mV, 0.9 +/- 0.2 ms), fast decay time constants (3.6 +/- 0.6 ms), and small amplitudes (-14 +/- 3.4 pA). Minimal electrical stimulation evoked monosynaptic EPSCs in only 48 +/- 9.2% of the trials suggesting that the CA3 pyramidal cell to basket cell pathway was unreliable. 3. CA3 pyramidal cell layer stimulation did not antidromically or synaptically activate granule cells but did evoke polysynaptic IPSCs in granule cells, suggesting that the net effect of CA3 pyramidal cell firing on the dentate gyrus was granule cell inhibition. 4. Stimulation of the CA3 pyramidal cell layer evoked both monosynaptic and polysynaptic EPSCs in basket cells, which were eliminated by a knife lesion separating CA3 from the dentate gyrus. The latencies of the EPSCs evoked in 0.6 mM extracellular calcium were the same as the earliest latencies of EPSCs in 1.5 mM calcium, suggesting that those EPSCs were monosynaptic. The polysynaptic input was more prominent in the presence of 10 microM bicuculline, implying that inhibitory GABAergic circuits normally limit this feedback from CA3 to basket cells. 5. In recordings from 103 pairs of CA3 pyramidal cells and dentate basket cells from 11 slices, two polysynaptic connections were found that were active only when the presynaptic CA3 pyramidal neuron fired in bursts. No monosynaptic connections between CA3 pyramidal cells and basket cells were identified indicating that connections between the two cell types may be sparse. 6. Raising the external potassium concentration from 3.5 to 8.5 mM, which elicited burst firing in CA3 pyramidal cells, resulted in a barrage of EPSCs and action potentials in basket cells. In contrast, granule cells neither fired action potentials nor exhibited increased EPSC frequency in elevated potassium but instead received a higher frequency of bicuculline-sensitive IPSCs, consistent with interneuron firing. The CA3 pyramidal cell to basket cell monosynaptic pathway exhibited paired-pulse facilitation as manifested by an increased probability of release, which supports the idea that basket cells were better activated by short trains of action potentials than by single inputs.(ABSTRACT TRUNCATED AT 400 WORDS)

Blind patch-clamp recordings from substantia gelatinosa neurons in adult rat spinal cord slices: Pharmacological properties of synaptic currents

Whole cell patch-clamp recordings were made from substantia gelatinosa neurons in the thick slice of the adult rat spinal cord, which retained an attached dorsal root to study the pharmacological properties of spontaneous and primary afferent fibre-evoked synaptic currents. The majority of substantia gelatinosa neurons tested exhibited miniature excitatory postsynaptic currents in the presence of tetrodotoxin (0.5 μM). Stimulation of primary afferent Aδ fibres evoked monosynaptic and/or polysynaptic excitatory postsynaptic currents. In Mg 2+-containing solution, 6-cyano-7-nitroquinoxaline-2,3-dione (10μM) abolished the evoked excitatory postsynaptic currents and the miniature excitatory postsynaptic currents. 2-Amino-5-phosphonovaleric acid (50–100μM) had little effect on the miniature excitatory postsynaptic current. In Mg 2+-free solution, however, 6-cyano-7-nitroquinoxaline-2,3-dione reduced but did not abolish the miniature excitatory postsynaptic currents, leaving the miniature excitatory postsynaptic currents with a small amplitude and a slow time course, which were abolished by 2-amino-5-phosphonovaleric acid. At holding potentials more positive than −60 mV, stimulation of Aδ fibres evoked outward postsynaptic currents in 11 out of 28 substantia gelatinosa neurons. The evoked inhibitory postsynaptic currents were abolished in seven out of 11 neurons by either strychnine (0.5μM) or bicuculline (10 μM), and in the remaining four neurons by the combination of both antagonists. These observations suggest that interneurons as well as Aδ fibres release l-glutamate, which evokes the excitatory postsynaptic current predominantly interacting with the non- N-methyl- d-aspartate receptor of the substantia gelatinosa neurons, and that the glycinergic and GABAergic inhibitory neurons are also likely to be involved in nociceptive transmission in the dorsal horn.

Ammonium ions abolish excitatory synaptic transmission between cerebellar neurons in primary dissociated tissue culture.

1. Transmitter glutamate is thought to be derived from glutamine via cleavage by glutaminase. NH+4 inhibits glutaminase. Therefore the decrease of glutamatergic excitatory synaptic transmission by NH+4 was thought to be due to the inability of glutamine to serve as precursor for glutamate. However, in cat spinal cord, NH+4 abolished excitatory synaptic transmission by a conduction block for action potentials in presynaptic terminals. The conduction block prevented inferences as to the effects of NH+4 on the availability of glutamate for synaptic transmission. This study reexamines the effects of NH+4 on glutamatergic excitatory synaptic transmission in cerebellar neurons in tissue culture. 2. Whole-cell patch voltage-clamp recordings were obtained from presumed Purkinje cells. Extracellular stimulation of presumed granule cells produced mono- and polysynaptic excitatory postsynaptic currents (EPSCs). In addition, presumed Purkinje cells showed spontaneous EPSCs that occurred independently of the addition of tetrodotoxin (TTX) or Cd2+ to the extracellular solution. 3. NH+4 (5-10 mM) abolished evoked mono- and polysynaptic EPSCs without abolishing spontaneous EPSCs and without significant effects on action currents in the Purkinje cell soma. 4. Increase of K+ in the extracellular solution to 10-12 from 5 mM abolished evoked EPSCs without abolishing spontaneous EPSCs and without significant effects on action currents in the Purkinje cell soma. 5. Mixtures of NH+4 and K+, with each ion in a concentration insufficient to affect evoked EPSCs when given alone, abolished evoked EPSCs when the sum of NH+4 and K+ exceeded 10-12 mM. 6. Increase of intracellular pH by trimethylamine had no effect on evoked and spontaneous EPSCs.(ABSTRACT TRUNCATED AT 250 WORDS)