Brain-derived neurotrophic factor promotes adaptive plasticity within the spinal cord and mediates the beneficial effects of controllable stimulation.

Brain-derived neurotrophic factor (BDNF) has been characterized as a potent modulator of neural plasticity in both the brain and spinal cord. The present experiments use an in vivo model system to demonstrate that training with controllable stimulation increases spinal BDNF expression and engages a BDNF-dependent process that promotes adaptive plasticity. Spinally transected rats administered legshock whenever one hind limb is extended (controllable stimulation) exhibit a progressive increase in flexion duration. This simple form of response-outcome (instrumental) learning is not observed when shock is given independent of leg position (uncontrollable stimulation). Uncontrollable electrical stimulation also induces a lasting effect that impairs learning for up to 48 h. Training with controllable shock can counter the adverse consequences of uncontrollable stimulation, to both prevent and reverse the learning deficit. Here it is shown that the protective and restorative effect of instrumental training depends on BDNF. Cellular assays showed that controllable stimulation increased BDNF mRNA expression and protein within the lumbar spinal cord. These changes were associated with an increase in the BDNF receptor TrkB protein within the dorsal horn. Evidence is then presented that these changes play a functional role in vivo. Application of a BDNF inhibitor (TrkB-IgG) blocked the protective effect of instrumental training. Direct (intrathecal) application of BDNF substituted for instrumental training to block both the induction and expression of the learning deficit. Uncontrollable stimulation also induced an increase in mechanical reactivity (allodynia), and this too was prevented by BDNF. TrkB-IgG blocked the restorative effect of instrumental training and intrathecal BDNF substituted for training to reverse the deficit. Taken together, these findings outline a critical role for BDNF in mediating the beneficial effects of controllable stimulation on spinal plasticity.

Intermittent noxious stimulation following spinal cord contusion injury impairs locomotor recovery and reduces spinal brain-derived neurotrophic factor-tropomyosin-receptor kinase signaling in adult rats.

Intermittent nociceptive stimulation following a complete transection or contused spinal cord injury (SCI) has been shown to exert several short- and long-lasting negative consequences. These include maladaptive spinal plasticity, enhanced mechanical allodynia, and impaired functional recovery of locomotor and bladder functions. The neurotrophin, brain-derived neurotrophic factor (BDNF) has been shown to play an important role in adaptive plasticity and also to restore functions following SCI. This suggests that the negative behavioral effects of shock are most likely related to corresponding changes in BDNF spinal levels. In this study, we investigated the cellular effects of nociceptive stimulation in contused adult rats focusing on BDNF, its receptor, tropomyosin-receptor kinase (TrkB), and the subsequent downstream signaling system. The goal was to determine whether the behavioral effect of stimulation is associated with concomitant cellular changes induced during the initial post-injury period. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to assess changes in the mRNA and/or protein levels of BDNF, TrkB, and the downstream signaling proteins calcium-calmodulin kinase II (CaMKII) and extracellular related kinase 1/2 (ERK1/2) at 1 h, 24 h, and 7 days following administration of intermittent noxious shock to the tail of contused subjects. In addition, recovery of locomotor function (Basso, Beattie, and Bresnahan [BBB] score) was assessed daily for the first week after injury. The results showed that, although nociceptive stimulation failed to induce any changes in gene expression at 1 h, it significantly reduced the expression of BDNF, TrkB, ERK2, and CaMKII at 24 h. In general, changes in gene expression were spatially localized to the dorsal spinal cord. In addition, locomotor recovery was impaired by shock. Evidence is also provided suggesting that shock engages a neuronal circuitry without having any negative effects on neuronal survival at 24 h. These results suggest that nociceptive activity following SCI decreases BDNF and TrkB levels, which may significantly contribute to diminished functional recovery.

Modulatory actions of serotonin, norepinephrine, dopamine, and acetylcholine in spinal cord deep dorsal horn neurons.

The deep dorsal horn represents a major site for the integration of spinal sensory information. The bulbospinal monoamine transmitters, released from serotonergic, noradrenergic, and dopaminergic systems, exert modulatory control over spinal sensory systems as does acetylcholine, an intrinsic spinal cord biogenic amine transmitter. Whole cell recordings of deep dorsal horn neurons in the rat spinal cord slice preparation were used to compare the cellular actions of serotonin, norepinephrine, dopamine, and acetylcholine on dorsal root stimulation-evoked afferent input and membrane cellular properties. In the majority of neurons, evoked excitatory postsynaptic potentials were depressed by the bulbospinal transmitters serotonin, norepinephrine, and dopamine. Although, the three descending transmitters could evoke common actions, in some neurons, individual transmitters evoked opposing actions. In comparison, acetylcholine generally facilitated the evoked responses, particularly the late, presumably N-methyl-D-aspartate receptor-mediated component. None of the transmitters modified neuronal passive membrane properties. In contrast, in response to depolarizing current steps, the biogenic amines significantly increased the number of spikes in 14/19 neurons that originally fired phasically (P < 0.01). Together, these results demonstrate that even though the deep dorsal horn contains many functionally distinct subpopulations of neurons, the bulbospinal monoamine transmitters can act at both synaptic and cellular sites to alter neuronal sensory integrative properties in a rather predictable manner, and clearly distinct from the actions of acetylcholine.

Serotonin 5-HT(2) receptor activation induces a long-lasting amplification of spinal reflex actions in the rat.

1. C-fibre activation induces a long-term potentiation (LTP) in the spinal flexion reflex in mammals, presumably to provide enhanced reflexive protection of damaged tissue from further injury. Descending monoaminergic pathways are thought to depress sensory input but may also amplify spinal reflexes; the mechanisms of this modulation within the spinal cord remain to be elucidated. 2. We used electrical stimulation of primary afferents and recordings of motor output, in the rat lumbar spinal cord maintained in vitro, to demonstrate that serotonin is capable of inducing a long-lasting increase in reflex strength at all ages examined (postnatal days 2-12). 3. Pharmacological analyses indicated an essential requirement for activation of 5-HT(2C) receptors while 5-HT(1A/1B), 5-HT(7) and 5-HT(2A) receptor activation was not required. In addition, primary afferent-evoked synaptic potentials recorded in a subpopulation of laminae III-VI spinal neurons were similarly facilitated by 5-HT. Thus, serotonin receptor-evoked facilitatory actions are complex, and may involve alterations in neuronal properties at both motoneuronal and pre-motoneuronal levels. 4. This study provides the first demonstration of a descending transmitter producing a long-lasting amplification in reflex strength, accomplished by activating a specific serotonin receptor subtype. It is suggested that brain modulatory systems regulate reflex pathways to function within an appropriate range of sensori-motor gain, facilitating reflexes in behavioural situations requiring increased sensory responsiveness.

Serotonin increases the incidence of primary afferent-evoked long-term depression in rat deep dorsal horn neurons.

5-hydroxytryptamine (5-HT) is released in spinal cord by descending systems that modulate somatosensory transmission and can potently depress primary afferent-evoked synaptic responses in dorsal horn neurons. Since primary afferent activity-induced long-term potentiation (LTP) may contribute to central sensitization of nociception, we studied the effects of 5-HT on the expression of sensory-evoked LTP and long-term depression (LTD) in deep dorsal horn (DDH) neurons. Whole cell, predominantly current clamp, recordings were obtained from DDH neurons in transverse slices of neonatal rat lumbar spinal cord. The effect of 5-HT on dorsal-root stimulation-evoked synaptic responses was tested before, during, or after high-frequency conditioning stimulation (CS). In most cells (80%), 5-HT caused a depression of the naïve synaptic response. Even though 5-HT depressed evoked responses, CS in the presence of 5-HT was not only still capable of inducing LTD but also increased its incidence from 54% in controls to 88% (P < 0.001). Activation of ligands selective for 5-HT(1A/1B) and 5-HT(1B), but not 5-HT(2A/2C) or 5-HT(3) receptors, best reproduced these actions. 5-HT also potently depressed postconditioning synaptic responses regardless of whether the induced plasticity was LTP or LTD. Our results demonstrate that in addition to depressing the amplitude of evoked sensory input, 5-HT can also control the direction of its long-term modifiability, favoring the expression of LTD. These findings demonstrate cellular mechanisms that may contribute to the descending serotonergic control of nociception.

Pharmacological characterization of serotonin receptor subtypes modulating primary afferent input to deep dorsal horn neurons in the neonatal rat.

Spinal cord slices and whole-cell patch clamp recordings were used to investigate the effects of serotonergic receptor ligands on dorsal root-evoked synaptic responses in deep dorsal horn (DDH) neurons of the neonatal rat at postnatal days (P) 3 - 6 and P10 - 14. Bath applied 5-hydroxytryptamine (5-HT) potently depressed synaptic responses in most neurons. Similarly, the 5-HT(1/7) receptor agonist, 5-carboxamidotryptamine (5-CT) depressed synaptic responses. This action was probably mediated by 5-HT(1A) receptor activation, since it occurred in the presence of the 5-HT(7) receptor antagonist clozapine and was not observed in the presence of NAN-190, a 5-HT(1A) receptor antagonist. In the absence of any agonist, 5-HT(1A) receptor antagonists often facilitated synaptic responses, suggesting that there is sufficient endogenous 5-HT to tonically activate 5-HT(1A) receptors. 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), the 5-HT(1A/7) receptor agonist, facilitated synaptic responses, an action probably mediated by 5-HT(7) receptors, since the facilitation could be reversed by subsequent application of the 5-HT(7) receptor antagonist clozapine. Agonists for the 5-HT(1B), 5-HT(2) and 5-HT(3) receptors exerted only modest modulatory actions. A pharmacological analysis of the depression evoked by 5-HT suggested an action partly mediated by 5-HT(1A) receptor activation, since antagonism of the 5-HT(1A) receptor with NAN-190 or WAY-100635 partly reversed 5-HT-evoked depression. In comparison, 5-HT(7) receptor activation could account for much of the 5-HT-evoked facilitation. We conclude that 5-HT is capable of modulating sensory input onto DDH neurons via several receptor subtypes, producing both facilitatory and depressant actions. Also, the actions of most receptor ligands on the evoked responses were similar within the first 2 postnatal weeks.

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.

Primary afferent-evoked synaptic plasticity in deep dorsal horn neurons from neonatal rat spinal cord in vitro.

Whole-cell patch clamp recordings of deep dorsal horn neurons were undertaken in 'thick' transverse slices to demonstrate plasticity of primary afferent-evoked synaptic responses following conditioning stimulation. Synaptic plasticity was observed in neurons throughout the age range examined (postnatal days 3-6 and 9-16) but only long-term depression (LTD) was evocable in older animals (P9-16). Both short- and long-latency synaptic responses could undergo long-term potentiation (LTP) and LTD suggesting that AMPA/kainate and NMDA receptor-evoked responses are modifiable.