Spinal Cord Injury Induces Changes in Electrophysiological Properties and Ion Channel Expression of Reticulospinal Neurons in Larval Lamprey

Animal care.

Larval sea lamprey [Petromyzon marinus (Pm)] were maintained in ∼10 L aquaria at 23–25°C and used for both neurophysiological and molecular biology experiments. The procedures used in the present study were approved by the Animal Care and Use Committee at the University of Missouri.

Spinal cord hemitransections.

Lamprey (n = 70 animals, ∼90–120 mm) were anesthetized in tricaine methanosulphonate (∼200 mg/L; MS222; Sigma, St. Louis, MO), and fine forceps and iridectomy scissors were used to make hemitransections (Fig. 1A, HT) on the right side of the spinal cord at 10% body length (normalized distance from the anterior head), ∼2 mm caudal to the brain at segment ∼2–3. In the lamprey, RS neurons are located in the mesencephalic reticular nucleus (MRN), as well as the anterior (ARRN), middle (MRRN), and posterior (PRRN) rhombencephalic reticular nuclei (Fig. 1B) (Rovainen, 1979). In the lamprey brain, large identified RS neurons, called “Müller cells,” have long ipsilateral axons that project to the caudal end of the spinal cord (Rovainen, 1979; Davis and McClellan, 1994b). The largest Müller cells in the MRRN are “B cells” (B1, B3, and B4), which were a major focus of the present study. In the MRRN and PRRN, ∼94 and ∼86%, respectively, of smaller unidentified RS neurons have ipsilateral descending axons (Shaw et al., 2001). Thus, hemitransections on the right side of the spinal cord axotomized all or most of the above neurons on the right side of the brain, whereas these neurons on the left side were mostly uninjured. In contrast, RS neurons in the ARRN have mixed ipsilateral/contralateral projections (Shaw et al., 2001), whereas <5% of the total number of RS neurons are located in the MRN (Davis and McClellan, 1994a).

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Figure 1.

A, Isolated brain/spinal cord preparation showing the brain (left) and rostral spinal cord, right spinal cord hemitransection (HT) at 10% body length (BL) (normalized distance from the anterior head), intracellular recording micropipette (IC), “rostral” suction electrode on the right, dorsal half of the spinal cord above the hemitransection, and “caudal” suction electrode around the spinal cord below the hemitransection. B, Diagram of the lamprey brain showing reticular nuclei: MRN, ARRN, MRRN, and PRRN (Rovainen, 1979). Large identified RS Müller cells (B cells; B1, B3, and B4) used in parts of the present study are located in the MRRN. Dotted lines indicate tissue excised for PCR experiments (see Materials and Methods).

Some animals had short recovery times (2–3 weeks; n = 50 animals), before substantial axonal regeneration has occurred (Davis and McClellan, 1994a,b) and at which time axotomy-induced changes in neurophysiological properties of RS neurons are most pronounced (McClellan et al., 2002; McClellan, 2003). Other animals had relatively long recovery times (12–16 weeks; n = 20 animals), at which time many axotomized RS neurons have regenerated their axons for at least 10 mm below the lesion (Davis and McClellan, 1994b), made synapse with spinal neurons (Mackler and Selzer, 1987), and display properties similar to those of uninjured neurons (McClellan et al., 2002).

Intracellular recordings.

After recovery, the brains and spinal cords, up to ∼30% body length, were removed, as described previously (Rouse et al., 1998), and pinned dorsal side up in a neurophysiological recording chamber containing oxygenated lamprey Ringer's solution (6–9°C; pH 7.4) (McClellan, 1990). A rostral suction electrode was placed on the right, dorsal surface of the spinal cord approximately halfway between the brain and spinal hemitransection, and a caudal suction electrode was placed around the spinal cord an average of 4.2 ± 3.7 mm (mean ± SD; n = 65 animals) or an average of ∼4.8 ± 4.2 segments below the hemitransections (Fig. 1A). Intracellular recordings using either current clamp (“bridge mode”) or “discontinuous current clamp” (DCC) (f_s ∼4–6 kHz) were made from uninjured and axotomized RS neurons with micropipettes filled with 5 m potassium acetate (∼60–100 MΩ). Only neurons with resting membrane potentials (V_rest) equal to or more negative than −65 mV were analyzed. There was no indication that axotomized RS neurons tended to have less negative membrane potentials than uninjured neurons. The effects of axotomy were examined in large identified RS neurons in the MRRN (B cells; B1, B3, and B4), as well as unidentified RS neurons in the MRRN and PRRN (Fig. 1B). The different B cells displayed similar changes in properties in response to axotomy. In the DCC mode, depolarizing pulses (+1–10 nA, 2 s) were applied to examine the effects of axotomy on repetitive firing patterns. In bridge mode, short 1–10 ms, +10 nA pulses were applied to elicit action potentials that were compared in uninjured and axotomized RS neurons. All data were stored on tape (11 kHz sampling rate per channel; Neuro-Data DR890; Cygnus Technologies, Delaware Water Gap, PA), as well as acquired by custom data acquisition and analysis software.

The action potential amplitude as well as the peak of the fast AHP (fAHP) (more negative than V_rest in ∼52% of uninjured B cells), afterdepolarizing potential (ADP) (more positive than V_rest in ∼75% of uninjured B cells), and slow AHP (sAHP) (always negative) were measured relative to resting membrane potential. If the peak of the fAHP was above or the peak of the ADP was below the resting potential (see Figs. 3C1, 4C1), values were not measured for these parameters. Because the fAHP, ADP, and sAHP occur in rapid succession, variations in the peaks of the fAHP and ADP (above or below V_rest) probably reflect slight differences in the amplitudes and/or timing of the three components of the afterpotential.

Orthodromic responses elicited by RS neurons were recorded rostral and caudal to the hemitransections (Fig. 1A). At short recovery times (2–3 weeks), the lack of orthodromic responses caudal to the hemitransections was taken to indicate that a given RS neuron, usually on the right side of the brain, had been axotomized. At long recovery times (12–16 weeks), orthodromic responses caudal to healed hemitransections elicited by axotomized RS neurons on the right side of the brain were taken as an indication that a neuron had regenerated its axon below the lesion site, at least for a few millimeters. Previous studies (Mackler and Selzer, 1987) indicate that, 7–40 weeks after spinal cord transection, stimulation of single Müller cells elicits ∼0.5–1.0 mV EPSPs in spinal neurons caudal to the lesion and does not elicit action potentials. In addition, the orthodromic responses are virtually identical before and after adding calcium channel blockers to the bath. Thus, these orthodromic responses, which were elicited at ∼1 Hz, are not attributable to activation of and recording from postsynaptic spinal targets.

In one group of control animals that did not receive hemitransections (n = 13 animals), the pharmacology of the sAHP in uninjured RS neurons was examined with one of the following agents added to the bath: 2 μm ω-conotoxin MVIIC (Tocris Bioscience, Ellisville, MO) to block high-voltage-activated (HVA) (N- and P/Q-type) calcium channels (McDonough et al., 1996; Bussieres and El Manira, 1999) or 20 μm apamin (Sigma) to block calcium-activated potassium (SKKCa) channels (Hill et al., 1992; Meer and Buchanan, 1992; Cangiano et al., 2002). Recordings were made at least 15 min after a drug was added to the bath. In a second group of control animals, firing patterns of uninjured RS neurons (n = 11 animals) were recorded before and after the addition of 2 mm CoCl₂ to the bath to block calcium influx and substantially reduce the sAHP to mimic the effects of axotomy (see Results).

Cloning lamprey ion channels.

In the present study, it was found that the sAHP is mediated by calcium influx via HVA calcium channels and activation of SKKCa channels and that the sAHP was significantly decreased after axotomy. Thus, the decreased sAHP might be attributable to downregulation of HVA calcium channels, SKKCa channels, or both. To distinguish these possibilities, we conducted experiments to measure mRNA expression levels for these channels in reticular nuclei containing uninjured and axotomized RS neurons.

We cloned and sequenced partial open reading frames for α subunits from two ion channels from the sea lamprey, Petromyzon marinus: an SKKCa channel and an HVA calcium channel (Ca_v2.x; see explanation below). This was accomplished using reverse transcription (RT)-PCR with a combination of both degenerate and specific oligonucleotide primers.

Ion channels were cloned from larval sea lamprey using RT-PCR with degenerate primers based on conserved amino acid and nucleotide alignments from Mus musculus, Rattus norvegicus, Homo sapiens, Danio rerio, and, when available, Tetraodon nigrividus. cDNA template derived from total RNA extracted from nervous system tissue (brain and spinal cord) was used to generate PCR amplicons of predicted size based on amino acid alignments. These PCR products were cloned into pGem-T-easy plasmid vector (Promega, Madison, WI) and sequenced using dye terminator cycle sequencing (CEQ DTCS Quick Start Kit; Beckman Coulter, Fullerton, CA). Sequences obtained were compared with orthologous sequences using BlastX (National Center for Biotechnology Information). GenBank accession numbers for these sequences are as follows (for Petromyzon marinus): PmHVA calcium, EF585497; PmSKKCa, EF585498.

The predicted amino acid sequence of our partial PmSKKCa open reading frame shares 90% amino acid identity with both human and mouse SKKCa channels. The predicted amino acid sequence of our partial HVA calcium channel sequence shares 79% amino acid identity (88% positives) with both P/Q-type (Ca_v2.1) and N-type (Ca_v2.2) calcium channels in humans and mice. Therefore, although we are confident in the identity of this as a HVA calcium channel (N- and P/Q-type but not L-type), we have not yet determined to which subfamily of HVA calcium channels our gene belongs. This was acceptable for the present study because the sAHP, which was substantially reduced in axotomized neurons, is primarily dependent on calcium influx via HVA channels (see Fig. 3D). Throughout this study, we refer to this channel as HVA calcium channels or PmCa_v2.x.

Real-time PCR.

Larval sea lamprey were anesthetized, spinal cord hemitransections were performed on the right side at 10% body length, and animals were allowed to recover for 1 week (n = 9 animals) or 11–17 weeks (n = 5 animals). A 1 week recovery time was used because it was assumed that molecular changes in ion channel expression would precede changes in biophysical properties, which are maximal at ∼2–3 weeks (McClellan et al., 2002). After recovery, the brains and rostral spinal cords were removed in lamprey Ringer's solution, and iridectomy scissors were used to make dorsoventral cuts in the brain to remove the MRRN and PRRN on the left and right sides of the brain (Fig. 1B, dotted lines). In larval sea lamprey (mean length, 125 mm), there are ∼80 (∼360) RS neurons in the MRRN (PRRN) on each side of the brain (Davis and McClellan, 1994a; Shaw et al., 2001; Zhang et al., 2002).

Each individual brain region was placed directly into 350 μl of lysis buffer (buffer RLT as provided by Qiagen, Valencia, CA) containing 1% β-mercaptoethanol and frozen at −80°C until RNA extraction. Primers specific for real-time PCR detection of PmSKKCa, PmCa_v2.x, and 18S rRNA (based on published sequence for lamprey; GenBank accession number M97575) using SYBR Green were developed and designed using Primer3 software. The sequences of primers were as follows: (1) 18S forward, 5′- CGACCGTTGCTTGGATAACT-3′; 18S reverse, 5′- CCGGATTGGTTTTGGTCTAA-3′; PmSKKCa forward, 5′- CACACACCTACTGTGGACGG-3′; (2) PmSKKCa reverse, 5′- GTTTCTCAGCCTTGGTCAGC-3′; and (3) PmCa_v2.x forward, 5′-ATTCTCGTCACGGAGCTTGG-3′; PmCa_v2.x reverse, 5′-GGAGGATGCGGATGGTGTAG-3′.

Total RNA was isolated from individual nuclei using the RNeasy Micro kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer, including an on-column DNase digestion to remove potential genomic DNA contamination. Reverse transcription was performed with SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) primed with a mix of both oligo-dT and random hexamers. cDNA was then purified via overnight ethanol precipitation and resuspended in nuclease-free water. Purified cDNA was used as a template in a PCR reaction in 1× Quantitect SYBR Green Master Mix (Qiagen) with 300 nm of each forward and reverse primers. PCR and fluorescence detection were performed in a RotorGene 3000 thermocycler (Corbett Research, Sydney, Australia) using 35–45 cycles as follows: 20 s at 95°C → 20 s at 58°C → 20 s at 72°C. Acquisition of SYBR Green fluorescence occurred at the 72°C step of the PCR and was analyzed with RotorGene 6 software (Corbett Research). Melt curves revealed only a single PCR product for any given reaction. Each reaction was performed in triplicate.

Care was taken at each step to minimize assay variability: samples were processed in parallel whenever possible, the same batch of reverse transcriptase was used for all samples, and PCR runs were designed to maximize the number of samples run in each batch. Fold difference in mRNA expression levels for HVA calcium or SKKCa channels between right and left nuclei in each animal then was calculated using the ΔΔCt method with 18S rRNA as the control gene for normalization (Livak, 1997).

Statistics.

For 2–3 week recovery times, neurophysiological parameters were determined for axotomized B cells (B1, B3, and B4 cells in the MRRN), which included all right B cells as well as those left B cells that did not elicit an orthodromic response in the recording electrode caudal to the hemitransections. If the ADP and sAHP components were clearly absent from the action potential (see Fig. 3B2), they were assigned values of zero. In other cases in which it was unclear whether a component was present or it was problematic to measure (e.g., fAHP peak above V_rest in Fig. 4C1; ADP peak below V_rest in Fig. 3C1), values were omitted from the analysis. Thus, in Table 1, there are sometimes different n values for the different parameters. Also, for some neurons, only firing patterns or electrophysiological parameters of action potentials were determined. ANOVA or Kruskal–Wallis test was used to compare the electrophysiological parameters between the following sets of B cells (Table 1): (1) uninjured neurons (always on left side) and axotomized neurons at short recovery times; (2) right and left neurons at long recovery times; and (3) left or right neurons at long recovery times and uninjured neurons at short recovery times. For the molecular data, the ratios of the mean fold levels of expression for left nuclei (containing mostly uninjured RS neurons; see above) and right nuclei (containing mostly axotomized RS neurons) for each cell group were compared with 1.0 (i.e., equal expression levels) using a one-sample t test. Statistical significance was assumed when p ≤ 0.05.