Characterization of calcium currents in functionally mature mouse spinal motoneurons

Motoneurons integrate synaptic input and produce output in the form of trains of action potentials such that appropriate muscle contraction occurs. Motoneuronal calcium currents play an important role in the production of this repetitive firing. Because these currents change in the postnatal period, it is necessary to study them in animals in which the motor system is 'functionally mature', that is, animals that are able to weight-bear and walk. In this study, calcium currents were recorded using whole-cell patch-clamp techniques from large (> 20 microm) ventral horn cells in lumbar spinal cord slices prepared from mature mice. Ninety percent (nine out of 10) of the recorded cells processed for choline acetyltransferase were found to be cholinergic, confirming their identity as motoneurons. A small number of motoneurons were found to have currents with low-voltage-activated (T-type) characteristics. Pharmacological dissection of the high-voltage-activated current demonstrated omega-agatoxin-TK- (P/Q-type), omega-conotoxin GVIA- (N-type), and dihydropyridine- and FPL-64176-sensitive (L-type) components. A cadmium-sensitive component of the current that was insensitive to these chemicals (R-type) was also seen in these cells. These results indicate that the calcium current in lumbar spinal motoneurons from functionally mature mice is mediated by a number of different channel subtypes. The characterization of these calcium channels in mature mammalian motoneurons will allow for the future study of their modulation and their roles during behaviours such as locomotion.     

The serotonin inhibition of high‐voltage‐activated calcium currents is relieved by action potential‐like depolarizations in dissociated cholinergic nucleus basalis neurons of the guinea‐pig

The aim of the present study was to investigate whether the voltage‐dependent inhibition of calcium currents by serotonin 5‐HT_1A agonists can be alleviated (facilitated) by action potential‐like depolarizations. In dissociated cholinergic basal forebrain neurons using whole‐cell recordings, it is shown that a selective serotonin 5‐HT_1A agonist (8‐OH‐DPAT) predominantly blocks N‐type HVA calcium current, although a minor reduction of P‐type current was also observed. The inhibition may principally occur through G_i‐G_o subtypes of G‐proteins because it was prevented by N‐ethylmaleimide, a substance known to block specifically pertussis‐sensitive G‐proteins. The inhibitory effect of 8‐OH‐DPAT on calcium currents is voltage‐dependent because it was alleviated by long‐lasting depolarizing prepulses. Interestingly, the inhibition could also be reversed by prepulses made‐up of action potential‐like depolarizations that were given at a frequency of 200 Hz. This observation may have important implications during periods of high‐frequency rhythmic bursts, a firing pattern that is prevalent in cholinergic basal forebrain neurons.     

Developmental regulation of T‐, N‐ and L‐type calcium currents in mouse embryonic sensory neurones

We investigated the development of a low (T-type) and two high voltage-activated (N- and L-type) calcium channel currents in large diameter dorsal root ganglion neurones acutely isolated from embryonic mice using the whole-cell patch-clamp technique. The low and high voltage-activated barium currents (LVA and HVA) were identified by their distinct threshold of activation and their sensitivity to pharmacological agents, dihydropyridines and ω-conotoxin-GVIA, at embryonic day 13 (E13), E15 and E17–18, respectively, before, during and after synaptogenesis. The amplitude and density of LVA currents, measured during a –40 mV pulse from a holding potential of –100 mV, increased significantly between E13 and E15, and remained constant between E15 and E17–18. The density of global HVA current, elicited by 0 mV pulse, increased between E13 and E15/E17–18. The density of the N-type current studied by the application of ω-conotoxin-GVIA (1 μm ) increased significantly between E13 and E15/E17–18. The use of the dihydropyridine nitrendipine (1 μm ) revealed that the density of L-type current remained constant at each stage of development. Nevertheless, application of dihydropyridine Bay K 8644 (3 μm ) demonstrated a significant slowing of the deactivation tail current between embryonic days 13 and 15, which may reflect a qualitative maturation of this class of calcium channel current. The temporal relationship between the changes in calcium channel pattern and the period of target innervation suggests possible roles of T-, N- and L-type currents during developmental key events such as natural neurone death and onset of synapse formation.     

Intracellular calcium stores modulate miniature GABA‐mediated synaptic currents in neonatal rat hippocampal neurons

The whole-cell configuration of the patch clamp technique was used to record miniature γ-aminobutyric acid_A (GABA_A) receptor-mediated currents (in tetrodotoxin, 1 μm and kynurenic acid 1 mm ) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6–9) old rats. Switching from a Ca²⁺-containing to a nominally Ca²⁺-free medium (in which Ca²⁺ was substituted with Mg²⁺, in the presence or in the absence of 100 μm EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration-dependent increase in frequency but not in amplitude of tetrodotoxin-resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 μm over control) was 1.8 ± 0.5, (n = 9). In nominally Ca²⁺-free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mm ), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mm ) reversibly reduced the amplitude of the current induced by GABA (100 μm ) indicating a clear postsynaptic effect on GABA_A receptor. Superfusion of ryanodine (30 μm ), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine–ryanodine-sensitive stores facilitates GABA release.     

Selective up-regulation of P- and R-type Ca2+ channels in rat embryo motoneurons by BDNF

Cultured spinal cord motoneurons from day 15 rat embryos (E15) represent a useful model to study Ca2+ channel diversities and their regulation by neurotrophins. Besides the previously identified L-, N- and P-type channels, E15 rat motoneurons also express high densities of R-type channels. We have previously shown that the P-type channel is nearly absent in 60% of these cells, while the R-type contributes to approximately 35% of the total current. Here, we show that chronic preincubation of cultured rat motoneurons with high concentrations (20-100 ng/mL) of brain-derived neurotrophic factor (BDNF) caused a selective up-regulation of the P- and R-type current density available after blocking N- and L-type channels, with no changes to cell membrane capacitance. N- and L-type channels were either not affected or slightly down-modulated by the neurotrophin. The onset of BDNF up-regulation of P/R-type currents had a half-time of 12 h and reached maximal values of approximately 80%. High concentrations of nerve growth factor (NGF; 50-100 ng/mL) had no effect on P/R currents, while BDNF action was prevented by the kinase inhibitor K252a and by the protein synthesis inhibitor anisomycin. These results suggest that chronic applications of BDNF selectively up-regulates the Ca2+ channel types which are most likely to be involved in the control of neurotransmitter release in mammalian neuromuscular junctions. The signal transduction mechanism is probably mediated by TrkB receptors and involves the synthesis of newly functionally active P- and R-type channels. Our data furnish a rationale for a number of recent observations in other laboratories, in which prolonged applications of neurotrophins were shown to potentiate the presynaptic response in developing synapses.     

European Neuroscience Association Pre‐ and post‐synaptic muscarinic receptors in thin slices of rat adrenal gland

The effects of activation of muscarinic receptors on chromaffin cells and splanchnic nerve terminals were studied in a rat adrenal slice preparation. In chromaffin cells, muscarine induced a transient hyperpolarization followed by a depolarization associated with cell spiking. The hyperpolarization was blocked by charybdotoxin (1 μm ) and tetraethylammonium chloride (TEA, 1 mm ), but was not affected by 200 μm Cd²⁺ or removal of external Ca²⁺, consistent with activation of BK channels. This would follow internal Ca²⁺ mobilization, as shown by Ca²⁺ imaging with fura-2 on isolated chromaffin cells in culture. Under voltage-clamp, outward BK currents were insensitive to MT3 toxin, a specific muscarinic m4 receptor antagonist. In contrast, muscarine-induced depolarization was due to a m4 receptor-mediated inward current blocked by MT3 toxin. This current was permeable to cations and was associated with Ca²⁺ entry and subsequently, Ca²⁺-induced Ca²⁺ release. Finally, both muscarine (25 μm ) and oxotremorine (10 μm ) decreased the amplitude and frequency of KCl-evoked excitatory postsynaptic currents, without affecting quantal size, consistent with a presynaptic inhibitory effect. Taken together, our data suggest that activation of m4 and probably m3 muscarinic receptors results in a strong, long-lasting excitation of chromaffin cells, as well as an uncoupling of synaptic inputs onto these cells.     

Embryonic rat motoneurons express a functional P-type voltage-dependent calcium channel

Only L- and N-type high voltage-activated calcium currents (HVA I_Ca) have been demonstrated in identified embryonic spinal motoneurons. However, pharmacological experiments suggest that other HVA I_Ca, including P-type, govern neurotransmitter release at the adult neuromuscular junction. We sought to analyse if embryonic motoneurons express these other I_Ca, using the whole-cell voltage-clamp method on motoneurons purified by a new metrizamide-panning technique from E15 rat embryos. In addition to L-type dihydropyridine-sensitive and N-type ω-GVIA-sensitive currents, motoneurons express two other HVA I_Ca. One has properties related to the P-type channel currents described in Purkinje cells: it is inhibited by the peptide ω-agatoxin-IVA with a maximal effect at 100–200 nM. The inhibited current has a characteristic sustained component during depolarizing test pulses. Furthermore, 50–100 nM concentration of ω-agatoxin-IVA reduce the increase in cytoplasmic calcium concentration observed after depolarization. The other HVA I_Ca is resistant to saturating concentrations of verapamil, ω-conotoxin GVIA and ω-agatoxin-IVA which block L, N and P-type HVAI_Ca, respectively. These results suggest that it is now possible to dissect, using a simple method of purification, the properties of the I_Ca in embryonic mammalian motoneurons and to provide pharmacological evidence for multiple calcium channels which may be involved in regulation of their activity during development.     

Dendritic L-type calcium currents in mouse spinal motoneurons: implications for bistability

The intrinsic properties of mammalian spinal motoneurons provide them with the capability to produce high rates of sustained firing in response to transient inputs (bistability). Even though it has been suggested that a persistent dendritic calcium current is responsible for the depolarizing drive underlying this firing property, such a current has not been demonstrated in these cells. In this study, calcium currents are recorded from functionally mature mouse spinal motoneurons using somatic whole-cell patch-clamp techniques. Under these conditions a component of the current demonstrated kinetics consistent with a current originating at a site spatially segregated from the soma. In response to step commands this component was seen as a late-onset, low amplitude persistent current whilst in response to depolarizing-repolarizing ramp commands a low voltage clockwise current hysteresis was recorded. Simulations using a neuromorphic motoneuron model could reproduce these currents only if a noninactivating calcium conductance was placed in the dendritic compartments. Pharmacological studies demonstrated that both the late-onset and hysteretic currents demonstrated sensitivity to both dihydropyridines and the L-channel activator FPL-64176. Furthermore, the alpha1D subunits of L-type calcium channels were immunohistochemically demonstrated on motoneuronal dendrites. It is concluded that there are dendritically located L-type channels in mammalian motoneurons capable of mediating a persistent depolarizing drive to the soma and which probably mediate the bistable behaviour of these cells.     

ω-Agatoxin IVA, a P-Type Calcium Channel Antagonist, Reduces Nociceptive Processing in Spinal Cord Neurons with Input From the Inflamed But Not From the Normal Knee Joint — An Electrophysiological Study in the Rat In Vivo

High threshold voltage-dependent P- and Q-type calcium channels are involved in neurotransmitter release. In order to investigate the role of P- and Q-type calcium channels in the mechanosensory (nociceptive) processing in the spinal cord, their participation in the responses of spinal wide-dynamic-range neurons to innocuous and noxious mechanical stimulation of the knee and ankle joints was studied in 30 anaesthetized rats. The knee was either normal or acutely inflamed by kaolin/carrageenan. During the topical application of ω-agatoxin IVA (P-type channel antagonist, 0.1 μM) onto the dorsal surface of the spinal cord, the responses to innocuous and noxious pressure applied to the normal knee were increased to respectively 124 ± 42% and 114 ± 23% of predrug values (mean ± SD, P < 0.05, 14 neurons). By contrast, in rats with an inflamed knee, the responses to innocuous and noxious pressure applied to the knee were reduced to respectively 72 ± 19 and 73 ± 22% of baseline (mean ± SD, P < 0.01, 13 neurons). In the same neurons, ω-agatoxin IVA slightly increased the responses to pressure on the non-inflamed ankle whether the knee was normal or inflamed. Thus P-type calcium channels seem to acquire a predominant importance in the excitation of spinal cord neurons by mechanosensory input from inflamed tissue and hence in the generation of inflammatory pain. By contrast, the Q-type channel antagonist, ω-conotoxin MVllC (1 or 100 μM), had no significant effect upon responses to innocuous or noxious pressure applied to either normal or inflamed knees (25 neurons).     

Modulation of calcium currents in mouse ventral horn neurons by extracellular pH

Neuronal activity has been shown to modulate the pH of the extracellular environment. Since neuronal circuits in the ventral horn of the spinal cord are highly active during patterned movements, and voltage-gated calcium channels play an important role in the production of spinal motoneuron output, the effects of changes in extracellular pH (pH(e)) on calcium currents in ventral horn neurons of the mouse spinal cord were examined. It is demonstrated that these channels are sensitive to modulation by pH(e). The amplitude of the current mediated by these channels increased as the pH(e) was elevated. The elevated pH(e) also led to a hyperpolarizing shift in the voltage dependence of both activation and inactivation. The opposite effects were seen for a decrease in pH(e). It was also noted that a decrease in pH(e) was associated with a faster inactivation of the current. It is concluded that voltage-gated calcium currents in ventral horn neurons are modulated by changes in pH(e), and that this modulation may play a physiologically important role in determining motoneuronal excitability during behaviors such as locomotion.     

Long‐term depression of C‐fibre‐evoked spinal field potentials by stimulation of primary afferent Aδ‐fibres in the adult rat

Long‐term potentiation (LTP) of spinal C‐fibre‐evoked field potentials can be induced by brief electrical stimulation of afferent C‐fibres, by natural noxious stimulation of skin or by acute nerve injury. Here, we report that in urethane anaesthetized, adult rats prolonged high frequency burst stimulation of the sciatic nerve at Aδ‐fibre strength produced long‐term depression (LTD) of C‐fibre‐evoked field potentials, and also depressed the increased amplitudes of C‐fibre‐evoked field potentials recorded after LTP had been established (depotentiation). Electrical stimulation of Aβ‐fibres failed to induce LTD or depotentiation. In spinalized rats, prolonged Aδ‐fibre conditioning stimulation induced LTP rather than LTD of C‐fibre‐evoked field potentials. Thus, tonic descending inhibition may determine the direction of plastic changes in C‐fibre‐mediated synaptic transmission. Spinal application of the N ‐methyl‐ d ‐aspartic acid receptor antagonist D‐APV blocked induction of LTD in intact rats and LTP in spinalized rats. The presently described LTD and the depotentiation of established LTP of C‐fibre‐evoked field potentials in spinal dorsal horn may underlie some forms of prolonged analgesia induced by peripheral nerve stimulation procedures.     

G‐proteins and G‐protein subunits mediating cholinergic inhibition of N‐type calcium currents in sympathetic neurons

One postsynaptic action of the transmitter acetylcholine in sympathetic ganglia is to inhibit somatic N-type Ca²⁺ currents: this reduces Ca²⁺-activated K⁺ currents and facilitates high-frequency spiking. Previous experiments on rat superior cervical ganglion neurons have revealed two distinct pathways for this inhibitory action: a rapid, voltage-dependent inhibition through activation of M₄ muscarinic acetylcholine receptors (mAChRs), and a slower, voltage-independent inhibition via M₁ mAChRs ( 1 –536]. We have analysed the mechanistic basis for this divergence at the level of the individual G-proteins and their α and βγ subunits, using a combination of site-directed antibody injection, plasmid-driven antisense RNA expression, over-expression of selected constitutively active subunits, and antagonism of endogenously liberated βγ subunits by over-expression of βγ-binding β-adrenergic receptor kinase 1 (βARK1) peptide. The results indicate that: (i) M₄ mAChR-induced inhibition is mediated by G_oA; (ii) α and βγ subunits released from the activated G_oA heterotrimer produce separate voltage-insensitive and voltage-sensitive components of inhibition, respectively; and (iii) voltage-insensitive M₁ mAChR-induced inhibition is likely to be mediated by the α subunit of G_q. Hence, Ca²⁺ current inhibition results from the concerted, but independent actions of three different G-protein subunits.     

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage‐sensitive calcium channels

The existence of voltage-sensitive Ca²⁺ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca²⁺ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca²⁺ concentration ([Ca²⁺]_i) variations induced by K⁺ depolarization and modified by specific Ca²⁺ channel agonists and antagonists. At rest, [Ca²⁺]_i was 90 ± 20 nm in both cell types. Microperifusion of high-K⁺ solution (50 mm ) for 1 s increased [Ca²⁺]_i to 290 ± 50 nm in type I (n = 20) and to 440 ± 50 nm in type II cells (n = 10). In Ca²⁺-free medium, K⁺ did not alter [Ca²⁺]_i. The specific L-type Ca²⁺ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K⁺-induced [Ca²⁺]_i increase in both cell types with maximum effect at 2 μm and 400 nm , respectively. Ni²⁺, a T-type Ca²⁺ channel blocker, reduced K⁺-evoked Ca²⁺ responses in a dose-dependent manner. For elevated Ni²⁺ concentrations, the response was differently affected by Ni²⁺ alone, or combined to nitrendipine (500 nm ). In optimal conditions, nitrendipine and Ni²⁺ strongly depressed by 95% the [Ca²⁺]_i increases. By contrast, neither ω-agatoxin IVA (1 μm ), a specific P- and Q-type blocker, nor ω-conotoxin GVIA (1 μm ), a specific N-type blocker, affected K⁺-evoked Ca²⁺_i responses. These results provide the first direct evidence that L- and probably T-type channels control the K⁺-induced Ca²⁺ influx in both types of sensory cells.     

High‐affinity copper block of GABAA receptor‐mediated currents in acutely isolated cerebellar Purkinje cells of the rat

The actions of Cu²⁺ ions on GABA_A receptor-mediated currents in acutely isolated Purkinje cells from rat cerebellum were studied using the whole-cell patch-clamp technique and a rapid perfusion system. Bath application of Cu²⁺ reduced currents induced by 2 μm γ-aminobutyric acid (GABA) in a concentration-dependent manner with an IC₅₀ of 35 n m . The Cu²⁺-induced block of GABA responses was not voltage-dependent. Increasing the GABA concentration (from 2 to 50 μm ) decreased the blocking effect of Cu²⁺. Dose–response analysis for activation of GABA_A receptors revealed a twofold decrease in apparent affinity for GABA in the presence of 0.1 μm Cu²⁺. Recovery from the block required several minutes after removal of Cu²⁺ from the medium. The block was removed by histidine, which preferentially forms complexes with Cu²⁺, or by other chelating substances. Application of 10 μm histidine immediately before application of 2 μm GABA completely relieved the block of GABA responses produced by 0.1 μm Cu²⁺. The effect of histidine was concentration-dependent with an EC₅₀ of 0.75 μm . The results demonstrate that Cu²⁺ is a potent inhibitor of GABA-evoked responses in rat Purkinje cells. Copper may be an endogenous synaptic modulating factor. Cu²⁺ toxicity, notably in Wilson’s disease, could result to some extent from chronic GABA_A receptor blockade.     

The voltage‐dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na⁺), calcium (Ca²⁺), and potassium (K⁺) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca²⁺-binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na⁺ current activated at about −45 mV, was half-maximal steady-state inactivated at −66.6 mV, and recovery from inactivation followed a two-exponential process (τ₁ = 8.4 ms and τ₂ = 858.8 ms). Na⁺ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (I_fast) activated ≈–40 mV, declined rapidly with a voltage-insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm ). I_fast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 μm ) and a second by high concentrations (6 mm ). After elimination of I_fast by a conditioning prepulse (50 ms to −50 mV), a slow K⁺ current (I_KV) could be studied in isolation. I_KV was only moderately affected by 4-AP (6 mm ), while TEA (20 mm ) blocked most (> 80%) of the current. I_KV activated at about −40 mV, declined monoexponentially in a voltage-dependent manner (τ≈ 850 ms at −30 mV), and revealed an incomplete steady-state inactivation. In addition to I_fast and I_KV, indications of a Ca²⁺-dependent outward current component were found. When Na⁺ currents, I_fast, and I_KV were blocked by tetrodotoxin (TTX, 1 μm ), 4-AP (6 mm ) and TEA (20 mm ) an inward current carried by Ca²⁺ was found. Ca²⁺ currents activated at depolarized potentials at about −30 mV, were completely blocked by 50 μm cadmium (Cd²⁺), were sensitive to verapamil (≈ 40% block by 10 μm ), and were not affected by nickel (50 μm ). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold).     

Expression of small‐conductance calcium‐activated potassium channels (SK) in outer hair cells of the rat cochlea

Physiological evidence suggests that SK-type Ca²⁺-activated K⁺ channels participate in ACh-induced hyperpolarization of OHCs (outer hair cells). Based on the sequences published by 1 , Science, 273 : 1709), we designed degenerated primers recognizing cDNA subunits of rSK1, rSK2 and rSK3. Using this concensus set of primers, we probed by PCR a rat organ of Corti cDNA library. Two PCR products of 707 base pairs with sequence identical to rSK3 and rSK2 were obtained and cloned to generate RNA probes for in situ hybridization in the rat cochlea. The subunit rSK2 showed hybridization in the organ of Corti, at the location of the OHCs. The expression of rSK2 by OHCs was confirmed by probing with PCR a poly(A) amplified OHC cDNA library. During development, rSK2 hybridization in the organ of Corti was negative at embryonic days E16, E18 and at P0, weak at P4 and stronger from P8 to adulthood. The subunit rSK2 could also be detected in the spiral ganglion from P4 to the adult stage. Contrary to rSK2, the subunit rSK3 did not show specific hybridization in the organ of Corti at the adult stage (P120) and only a weak expression was observed at P10 and P21. Our study demonstrates expression of rSK2 in OHCs. These potassium channels are good candidates to underlie the ACh-activated K⁺ currents recorded during patch-clamp recordings in isolated OHCs. The expression of rSK2 in the cochlear ganglion at the adult stage suggests that SK Ca²⁺-activated K⁺ channels may also participate in the repolarization of the auditory neurons after the action potential and may influence their firing patterns.     

VAMP‐1 and VAMP‐2 gene expression in rat spinal motoneurones: differential regulation after neuronal injury

Vesicle-associated membrane protein (VAMP; synaptobrevin) is involved in the molecular regulation of transmitter release at the presynaptic plasma membrane. VAMP exists in two isoforms, VAMP-1 and VAMP-2, which are transcribed from two separate genes and differentially expressed in the nervous system. In situ hybridization was used to examine whether VAMP isoform mRNA expression may be altered by experimental manipulations. The effect of nerve injury on VAMP-1 and VAMP-2 mRNA levels in motoneurones of the rat lumbar spinal cord was compared with lesion-induced changes in the expression of choline acetyl transferase (ChAT) and α-calcitonin gene-related peptide (α-CGRP) mRNA. After unilateral sciatic nerve transection (axotomy), VAMP-1 mRNA expression decreased significantly in parallel with a downregulation of ChAT mRNA in axotomized motoneurones compared with the corresponding motoneurones on the contralateral unlesioned side. There was a rapid decrease in VAMP-1 and ChAT mRNA levels at 2 days after axotomy, and at 7 days there was a 65% decrease in VAMP-1 mRNA and a 48% decrease in ChAT mRNA. VAMP-1 mRNA levels continued to decrease at 14 and 21 days, while ChAT mRNA levels had returned to normal at this time. In contrast, VAMP-2 and α-CGRP mRNA levels were upregulated in axotomized motoneurones. A significant increase for both VAMP-2 and α-CGRP mRNA levels was present 2 days after axotomy, and a maximum was reached after 7 days for α-CGRP mRNA (163%) and after 14 days for VAMP-2 mRNA (587%). Immunohistochemical analysis did not reveal any detectable changes in VAMP-1- or VAMP-2-like immunoreactivity in the motoneurone cell soma after axotomy. In the proximal end of the transected sciatic nerve, there was an increase in VAMP-1- and VAMP-2-LI, which was most prominent at 2 days after lesion. The results show that, in axotomized spinal motoneurones, VAMP-1 mRNA is downregulated and VAMP-2 mRNA is upregulated, indicating differential regulation of the two separate VAMP genes and differential roles for the two VAMP isoforms in the regulation of exocytosis after nerve injury.