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Serotonergic modulation of nociceptive circuits in spinal cord dorsal horn
Full text linkBackground: Despite the extensive number of studies performed in the last 50 years, aimed at describing the role of serotonin and its receptors in pain modulation at the spinal cord level, several aspects are still not entirely understood. The interpretation of these results is often complicated by the use of different pain models and animal species, together with the lack of highly selective agonists and antagonists binding to serotonin receptors. Method: In this review, a search has been conducted on studies investigating the modulatory action exerted by serotonin on specific neurons and circuits in the spinal cord dorsal horn. Particular attention has been paid to studies employing electrophysiological techniques, both in vivo and in vitro. Conclusion: The effects of serotonin on pain transmission in dorsal horn depend on several factors, including the type of receptors activated and the populations of neurons involved. Recently, studies performed by activating and/or recording from identified neurons have importantly contributed to the understanding of serotonergic modulation on dorsal horn circuits
Experimental Protocols and Analytical Procedures for Studying Synaptic Transmission in Rodent Spinal Cord Dorsal Horn
No full textSynaptic modulation and plasticity are key mechanisms underlying pain transmission in the spinal cord and supra-spinal centers. The study and understanding of these phenomena are fundamental to investigating both acute nociception and maladaptive changes occurring in chronic pain. This article describes experimental protocols and analytical methods utilized in electrophysiological studies to investigate synaptic modulation and plasticity at the first station of somatosensory processing, the spinal cord dorsal horn. Protocols useful for characterizing the nature of synaptic inputs, the site of modulation (pre- versus postsynaptic), and the presence of short-term synaptic plasticity are presented. These methods can be employed to study the physiology of acute nociception, the pathological mechanisms of persistent inflammatory and neuropathic pain, and the pharmacology of receptors and channels involved in pain transmission. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Spinal cord dissection and acute slice preparation Basic Protocol 2: Stimulation of the dorsal root and extracellular recording (compound action potentials and field potentials) Basic Protocol 3: Patch-clamp recording from dorsal horn neurons: action potential firing patterns and evoked synaptic inputs Basic Protocol 4: Analysis of parameters responsible for changes in synaptic efficacy Basic Protocol 5: Recording and analysis of currents mediated by astrocytic glutamate
Modifications of A-current kinetics in mammalian central neurones induced by extracellular zinc.
No full text1. Whole-cell voltage clamp recordings were used to study the action of the transition ion zinc on the A-current kinetics in granule cells from rat cerebellar slices. 2. The effects of zinc have been tested in the concentration range from 1 microM to 1 mM, and fully characterized on all kinetic parameters at 100 and 300 microM. All the effects observed were rapid, concentration dependent and fully reversible. 3. Steady-state inactivation curves are strongly shifted towards depolarized potentials, with activation curves much less so. These shifts lead to an increase of the peak current amplitude around physiological resting membrane potentials and to a decrease at hyperpolarized potentials. 4. The forward 'on' rate constants are slowed by Zn2+ at a concentration of 100-300 microM by a factor from 1.5 to 4. The backward 'off' rate constants are unaffected by Zn2+. 5. The development of IA inactivation, as measured from the current decay, is not affected by Zn2+ up to 1 mM. Removal of inactivation is, on the contrary, significantly slowed. 6. The results are neither compatible with the theory of the surface charge screening effect nor with a mechanism involving channel block. It seems more likely that Zn2+ interferes with the channel gating by binding to a specific domain of the channel protein. 7. After treatment with Hg2+, which is irreversible, Zn2+ still maintains its effects, which suggest that the two divalents act at different sites. 8. In view of the widespread distribution of zinc throughout the brain, its actions on the A-current could play an important role in physiological function
Kinetic analysis of a transient potassium current in rat cerebellar granule cells in vitro
No full textThin slices were prepared from cerebella of 10-24 day old rats and examined with whole-cell patch-clamp methods. Depolarizing steps from holding potentials negative to -60 mV elicited an early transient outward current, identified as IA, and a late outward K+ current. Depolarizations from -50 mV failed to evoke any A current and gave only a slowly rising component similar to the delayed K+ current, which inactivated thereafter with a time constant of 2.5 s at -30 mV. The IA peaked in 1-2 ms, decayed following a double exponential with time constants of 8.1 and 53.2 ms at +20 mV and was half-inactivated at -82.5 mV. 4-AP 4 mM depressed both K+ currents showing little specificity between them, while TEA 20 mM selectively abolished only the delayed K+ current
BDNF and TrkB mediated mechanisms in the spinal cord
No full textThe neurotrophin brain-derived neurotrophic factor (BDNF) plays an essential role during development, promoting the survival of specific populations of central and peripheral neurons. During adulthood, BDNF also acts as a synaptic modulator in several areas of the central nervous system (CNS), including the spinal cord, and is involved in short and long term changes of synaptic efficacy. In spinal cord dorsal horn BDNF is expressed in the peptidergic terminals originating from primary afferent fibres, while its high affinity receptor trkB has been detected on both primary afferent terminals and dorsal horn neurons. In superficial dorsal horn, exogenous BDNF modulates fast excitatory (glutamatergic) and inhibitory (GABAergic/glycinergic) signals, as well as slow peptidergic neurotransmission. Conditions of inflammatory and neuropathic pain alter the expression of BDNF and trkB receptors in dorsal horn. In experimental pain models, modulation of synaptic transmission by BDNF plays an important role in the induction and maintenance of central sensitization
Modulating action of extracellular zinc on transient potassium currents in cerebellar granule cells in the rat
No full textThe effect of Zn2+ 100 microM-1 mM has been studied on the kinetics of the A-current in granule cells from rat cerebellar slices using the patch-clamp technique in the whole-cell configuration. Zn2+ induced marked shifts towards positive potentials of both the activation and inactivation steady-state curves, a reduction of maximal amplitude and a slowing of the activation kinetics, leaving unaffected the inactivation time constants. These modifications cannot be explained in terms of the screening of the negative surface charges, but are probably due to a direct action on the A-channel. The alterations observed in the IA kinetics could be of physiological relevance in some neurological disorders for which significant increase of the Zn2+ levels in the cerebrospinal fluid have been described
Kinetic study and numerical reconstruction of A-type current in granule cells of rat cerebellar slices
No full text1. Whole-cell voltage-clamp techniques were used to study voltage-activated transient potassium currents in a large sample (n = 143) of granule cells (GrC) from rat cerebellar slices. Tetrodotoxin (TTX; 0.1 microM) was used to block sodium currents, while calcium current was too small to be seen under ordinary conditions. 2. Depolarizing pulses from -50 mV evoked a slow, sustained outward current, developing with a time constant of 10 ms, inactivating over a time scale of seconds and which could be suppressed by 20 mM tetraethylammonium (TEA). By preventing the Ca2+ inflow, this slow outward current could be further separated into a Ca(2+)-dependent and a Ca(2+)-independent component. 3. After conditioning hyperpolarizations to potentials negative to -60 mV, depolarizations elicited transient outward current, peaking in 1-2 ms and inactivating rapidly (approximately 10 ms at 20 degrees C), showing the overall kinetic characteristics of the A-current (IA). The current activated following third-order kinetics and showed a maximal conductance of 12 nS per cell, corresponding to a normalized conductance of 3.8 nS/pF. 4. IA was insensitive to TEA and to the Ca(2+)-channel blockers. 4-Aminopyridine (4-AP) reduced the A-current amplitude by approximately 20%, and the delayed outward currents by > 80%. 5. Voltage-dependent steady-state inactivation of peak IA was described by a Boltzmann function with a slope factor of 8.4 mV and half-inactivation occurring at -78.8 mV. Activation of IA was characterized by a Boltzmann curve with the midpoint at -46.7 mV and with a slope factor of 19.8 mV. 6. IA activation and inactivation was best fitted by the Hodgkin-Huxley m3h formalism. The rate of activation, tau a, was voltage-dependent, and had values ranging from 0.55 ms at -40 mV to 0.2 ms at +50 mV. Double-pulse experiment showed that development and removal of inactivation followed a single-exponential time course; the inactivation time constant, tau ha, was markedly voltage-dependent and ranged from approximately 10 ms at -40 and -100 mV and 70 ms at -70 mV. 7. A set of continuous equations has been developed describing the voltage-dependence of both the steady-state and time constant of activation and inactivation processes, allowing a satisfactory numerical reconstruction of the A-current over the physiologically significant membrane voltage range.(ABSTRACT TRUNCATED AT 400 WORDS
Serotonergic modulation by 5-HT7 receptors in mouse spinal cord dorsal horn
No full textBackground
Serotonergic receptors of the 5-HT7 type (5-HT7Rs) are widely expressed in the central nervous system, where they modulate seve ral functions, such as sleep induction, learning, mood, and vegetative behaviours. Along the pain axis, 5-HT7Rs are expressed on
nociceptive primary afferent fibers and in the dorsal horn, both on neurons and astrocytes. [1]. Behavioural experiments have produ ced controversial results about anti- and pro-nociceptive actions of 5-HT7Rs. The low agonist selectivity and the different pain animal
models used have likely contributed to the heterogeneity of the results [2].
To investigate the effects of 5-HT7Rs on spinal pain, we have performed an electrophysiological study on mouse spinal cord slices,
using the selective agonist LP-211 [3]. The recorded neurons have been functionally characterized, in order to identify the neural
circuits involved in the serotonergic modulation.
Methods
Patch-clamp recording was performed on lamina II neurons in spinal cord slices obtained from postnatal CD1 mice (P15-P25) [4]. Excitatory
postsynaptic currents (EPSCs) were recorded in voltage clamp; evoked EPSCs were elicited by stimulating the dorsal root with a suction
electrode in the Aδ and C fibers range.
Results
Application of 1 μM LP-211 to the spinal cord slice induced a facilitation of glutamatergic transmission: the frequency of spontaneous
EPSCs was significantly increased in a subpopulation of neurons (control: 0.9±0.2 Hz; LP-211: 1.8±0.6 Hz; 5 responsive neurons out of
8). The recorded neurons were characterized from their firing pattern: significant effects of LP-211 were observed in both tonic and de layed firing neurons, corresponding to inhibitory and excitatory interneurons, respectively.
Application of 1 μM LP-211 in the presence of 10 μM SB269970 (a 5-HT7R antagonist) did not alter spontaneous EPSC frequency in 11
lamina II neurons, confirming the involvement of 5-HT7Rs in glutamate release facilitation.
EPSCs evoked by dorsal root stimulation were also tested with LP-211. The currents, evoked by paired pulse protocol, were significantly
potentiated by the compound (mean potentiation: 19±4.2%; 4 responsive neurons out 7). The second EPSC was less potentiated than
the first and the paired pulse ratio decreased in 3 neurons.
Conclusion
The compound LP-211 is able to selectively activate 5-HT7Rs in the dorsal horn, causing a facilitatory effect of both spontaneous and
evoked EPSCs. The decrease of paired-pulse ratio suggests that LP-211 activates presynaptic 5-HT7Rs, increasing glutamate release.
The study of specific effects of these receptors on the different neuron populations will be critical to determine whether 5-HT7Rs exert
anti- or pro-nociceptive effects at the spinal level.
References
1. Cortes-Altamirano JL et al., Curr. Neuropharm, 2018, 16:210-221
2. Bardoni R, Curr. Neuropharm, 2019, 17:1133-1145
3. Hedlund PB et al, Neurosci Lett. 2010 481:12-6.
4. Betelli C et al., Mol. Pain, 2015, 11:
Excitatory synapses in the glomerular triad of frog olfactory bulb in vitro
No full textWhole-cell patch clamp recording techniques were applied to periglomerular (PG) cells in slices of the frog olfactory bulb (OB) to study the properties of the excitatory synapses in the triad formed by the olfactory nerve (ON) and the dendrites of mitral/tufted (MT) cells and PG cells. The postsynaptic response evoked by ON stimulation was glutamatergic and could be dissected into NMDA and non-NMDA components of equivalent amplitudes. The dendro-dendritic synapse between MT and PG cells could be activated following antidromic stimulation of the lateral and medial olfactory tract (LOT and MOT). In this case the postsynaptic potentials had amplitudes and durations comparable to those obtained by ON stimulation, the neurotransmitter was glutamate, but the synapse was largely dominated by the slow NMDA component
Mechanisms of Peripheral and Central Pain Sensitization: Focus on Ocular Pain
Full text linkPersistent ocular pain caused by corneal inflammation and/or nerve injury is accompanied by significant alterations along the pain axis. Both primary sensory neurons in the trigeminal nerves and secondary neurons in the spinal trigeminal nucleus are subjected to profound morphological and functional changes, leading to peripheral and central pain sensitization. Several studies using animal models of inflammatory and neuropathic ocular pain have provided insight about the mechanisms involved in these maladaptive changes. Recently, the advent of new techniques such as optogenetics or genetic neuronal labelling has allowed the investigation of identified circuits involved in nociception, both at the spinal and trigeminal level. In this review, we will describe some of the mechanisms that contribute to the perception of ocular pain at the periphery and at the spinal trigeminal nucleus. Recent advances in the discovery of molecular and cellular mechanisms contributing to peripheral and central pain sensitization of the trigeminal pathways will be also presented
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