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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Behav Pharmacol. 2021 Jun 1;32(4):259–264. doi: 10.1097/FBP.0000000000000608

Acute Intrathecal Administration of Quipazine Elicits Air-Stepping Behavior

Hillary E Swann-Thomsen 1, Derek D Viall 2, Michele R Brumley 3
PMCID: PMC8119288  NIHMSID: NIHMS1635992  PMID: 33595953

Abstract

Serotonin plays a pivotal role in the initiation and modulation of locomotor behavior in the intact animal, as well as following spinal cord injury. Quipazine, a serotonin 2 receptor agonist, has been used successfully to initiate and restore motor behavior in rodents. Although evidence suggests that the effects of quipazine are spinally mediated, it is unclear whether intrathecal (IT) quipazine administration alone is enough to activate locomotor-like activity or whether additional stimulation is needed. Thus, the current study examined the effects of IT administration of quipazine in postnatal day 1 rats in two separate experiments. In Experiment 1, quipazine (0.1, 0.3, or 1.0 mg/kg) was dissolved in saline and administered via IT injection to the thoracolumbar cord. There was no significant effect of drug on hindlimb alternating stepping. In Experiment 2, quipazine (0.3 or 1.0 mg/kg) was dissolved in a polysorbate 80-saline solution (Tween 80) and administered via IT injection. Polysorbate 80 was used to disrupt the blood brain barrier to facilitate absorption of quipazine. The injection was followed by tail pinch 5 minutes post-injection. A significant increase in the percentage of hindlimb alternating steps was found in subjects treated with 0.3 mg/kg quipazine, suggesting that IT quipazine when combined with sensory stimulation to the spinal cord, facilitates locomotor-like behavior. These findings indicate that dissolving the drug in polysorbate 80 rather than saline may heighten the effects of IT quipazine. Collectively, this study provides clarification on the role of quipazine in evoking spinally-mediated locomotor behavior.

It is well known that serotonin plays an important role in the initiation and modulation of locomotion (Schmidt & Jordan, 2000; Sławińska, Miazga, & Jordan, 2014), including during early development (Brumley & Robinson, 2005; Swann et al., 2016) and following spinal cord injury (for review see Ghosh & Pearse, 2015). For instance, in developing rats, hindlimb locomotor-like stepping behavior following systemic administration of a serotonergic agonist (quipazine) has been observed following a spinal transection that isolates lower (i.e., hindlimb) spinal circuits from more rostral influences (Brumley & Robinson, 2005; McEwen et al., 1997; Strain et al., 2014). In particular, much evidence suggests that 5-HT2 receptors play a key role in mediating locomotor responses at the level of the spinal cord (Liu & Jordan, 2005; Ung et al., 2008), including below the site of a spinal injury (Fouad et al., 2010; Sławińska, Miazga, & Jordan, 2014).

Quipazine is a 5-HT2 receptor agonist that has been used in numerous studies investigating the spinal control or restoration of locomotion (e.g., Antri et al., 2003; Landry & Guertin, 2004; Ung 2008), and often may be coupled with sensory (Dugan & Shumsky, 2015; Fong et al., 2005; Sławińska, Majczyński, Dai, & Jordan, 2012; Swann et al., 2017) or epidural spinal cord stimulation (Chia et al., 2020; Gerasimenko et al., 2007) to induce and/or improve stepping behavior in spinal-injured animals. Although evidence suggests that quipazine’s actions are centrally-mediated and likely involve direct activation of locomotor networks in the spinal cord (Cowley et al., 2015; Oueghlani et al., in press; Ziegler et al., 2011), it also is difficult to parse out whether or not intrathecal (IT) quipazine administration directly activates locomotor-like activity in vivo without the influence of step training. For instance, one study found that IT quipazine administration alone did not improve step behavior without being combined with robotic-assisted treadmill training, in spinal transected adult rats (de Leon & Acosta, 2006). Thus, although quipazine is largely believed to be acting at the level of the spinal cord, clear evidence that it can evoke locomotor behavior following direct spinal application remains elusive.

Therefore, in the present study we evaluated the effects of acute IT quipazine administration on locomotor stepping behavior in the rat neonate. We used the newborn rat air-stepping model, in which systemic (intraperitoneal) quipazine has been shown to induce robust alternating stepping of the limbs (Strain & Brumley, 2014; Swann et al., 2016) within 5 min, including of the hindlimbs following complete mid- or low-thoracic spinal cord transection (McEwen et al., 1997; Strain et al., 2014). In this model, newborn rats are suspended in the air using a harness, whereby their limbs hang freely and unimpeded; meanwhile, the need for postural control is alleviated (Brumley, Guertin, & Taccola, 2017). Here, two experiments were conducted to determine if IT administration of quipazine would evoke alternating stepping of the hindlimbs in the intact animal, when the drug was applied in the area of thoracolumbar (hindlimb) locomotor circuits (Cowley & Schmidt, 1997) activated by serotonin.

In the first experiment, newborn rats received one of three doses of quipazine or saline (vehicle control) via IT injection at the low thoracic level of the spinal cord. In a follow-up second experiment, quipazine was dissolved in a Tween 80 solution (vehicle control) and followed by a tail pinch (to increase excitatory drive to locomotor centers). It was hypothesized that IT administration of quipazine would induce stepping behavior in both experiments. Results have important implications for interpreting experiments using quipazine in models of spinal cord injury and recovery.

General Methods

Subjects

Subjects were male and female newborn offspring of Sprague-Dawley rats time-mated in the Animal Care Facility at Idaho State University. Newborn subjects stayed in their home cage with the dam until the day of testing. Animals were maintained on a 12:12 h light-dark cycle, with food and water available ad libitum. All procedures involving the care and use of animals were reviewed and approved by the university Institutional Animal Care and Use Committee and were in accordance with NIH Guidelines (National Research Council, 2011).

A total of 56 postnatal day 1 (P1; ~24 h after birth) rat pups were used as subjects across two experiments. Prior to testing, subjects were examined for signs of recent feeding, evidenced by the presence of a milk band on the abdomen, and overall, were in good health. Subjects were tested in one experiment only. In order to avoid litter effects, no more than one subject per condition was used from a single litter. Following testing, subjects were euthanized humanely.

Experimental Set-Up

Rat pups were individually tested inside an infant incubator that regulated humidity and maintained air temperature at 35°C. Immediately prior to testing, subjects were manually voided, and body mass was recorded. Then subjects were placed inside the incubator to acclimate to testing conditions. During acclimation, subjects remained inside a small, plastic bowl with a couple of siblings.

Following 30 min of acclimation, subjects received an intrathecal (IT) injection of quipazine or vehicle control (described below). Subjects were then gently secured to a rubber-covered horizontal bar in the prone posture, as is typical in air-stepping experiments with newborn rats (i.e., using a harness that supports the head and body but does not interfere with limb movements and limbs hanging pendant) (Swann et al., 2016). Behavior was recorded for a 20-min test session post-injection, which was captured by a micro-camera located inside the incubator and connected to an outside recording DVD unit. The micro-camera was positioned below the subjects, permitting a clear view of all limbs. SMPTE time-code was impressed on video recordings throughout the test session.

Intrathecal Injection

IT injections (1 μL) were delivered between T11 and T12 vertebrae of the spinal cord with a 30-gauge needle. The needle was connected to Polyethelene (PE-10) tubing which was pre-loaded with drug or vehicle solution, depending on testing condition. The other end of the tubing was connected to an automated infusion pump. To begin the injection, the needle was quickly guided directly into the intrathecal space of the pup by an experimenter (blinded to drug condition), using landmarks on the back of the subject. (Pilot experiments using blue dye indicated that the landmark used was associated with the low thoracic region, with 1 μL IT injection of dye spreading between T10 and L1.) A second experimenter activated the infusion pump, and IT infusion of the drug occurred in a 3-sec pulse. During the procedure, the first experimenter continued to gently hold the subject until the injection was complete. Although subjects briefly responded to the initial needle poke, they remained relatively quiet throughout the remainder of the quick procedure. Subjects were then placed on the horizontal bar for the 20-min test session to evaluate stepping behavior.

Behavioral Scoring of Hindlimb Activity

The 20-min test session was recorded and later scored during video playback at normal or reduced speed. Events were entered into a computer using an event recorder program (JWatcher) to record the category of behavior and time of entry (+/− 0.01s). Behaviors that were scored included hindlimb alternating steps, synchronous donkey kicks, and individual hindlimb movements. As donkey kicks occurred very infrequently, we did not analyze them further. Alternating steps consisted of consecutive and opposing extension and flexion of the two hindlimbs (Brumley, Roberto, & Strain, 2012). All other limb activity was categorized as non-stepping movement. During scoring, the experimenter was blind to the subject’s testing (drug) condition. Intra- and interrater reliability for scoring was >90%.

Data Analysis

Analyses were conducted separately for Experiments 1 and 2. Because we were interested in the effect of drug condition on stepping behavior, we analyzed frequency of hindlimb alternating steps and percentage of hindlimb alternating steps (as function of number of total limb movements) in each experiment. Behavior was summed across the 20-min test session, and analyzed using one-way ANOVAs. The independent variable in each ANOVA was drug condition and the dependent variables were alternating step frequency and percentage of alternating steps. Tukey’s post-hoc comparisons of means were conducted based on significant main effects. IBM SPSS Statistical software was used for analysis, and a 5% significance level was adopted for all tests.

Experiment 1: Effect of Acute IT Quipazine Administration on Stepping Behavior, Using Saline as Vehicle Control

Newborn rats show robust alternating air-stepping behavior following intraperitoneal (IP) administration of quipazine (e.g., Strain & Brumley, 2014; Swann et al., 2016). The vehicle control used for diluting and delivering quipazine in these experiments has been saline, with a dose of 3.0 mg/kg determined to be the most effective dose for eliciting stepping. Because IP injection of quipazine elicits hindlimb stepping in rodents that have received a mid-thoracic spinal cord transection (Strain et al., 2014; Ung et al., 2008), it is thought that quipazine activates spinal cord circuitry. To answer this question more clearly, here we delivered direct spinal administration of quipazine, via intrathecal injection, to the region of the spinal cord that is involved with producing hindlimb locomotor activity (Cowley & Schmidt, 1997). The purpose of the experiment was to examine the effects of IT administration of quipazine on alternating hindlimb stepping behavior in newborn rats, using the air-stepping paradigm.

Experimental Design

A total of 32 P1 rats (16 males, 16 females) were used as subjects in Experiment 1. Subjects were assigned to one of four drug conditions: 0.1 mg/kg Quipazine, 0.3 mg/kg Quipazine, 1.0 mg/kg Quipazine, or saline (vehicle control). Subjects received an IT injection of 1 μL of solution at the low thoracic spinal level, were placed in the harness on the bar, and then had their behavior recorded for 20-min as described above.

Pharmacology

Quipazine maleate, a serotonin receptor agonist (Sigma-Aldrich, St. Louis, MO) was dissolved in 0.9% saline and prepared in doses of 0.1 mg/kg, 0.3 mg/kg, or 1.0 mg/kg. Studies with adult rats that have administered quipazine intrathecally (Ziegler et al., 2010) used doses that were 1/10th of the dose used intraperitoneally (Ichiyama et al., 2008; Lavrov et al., 2008). Therefore, the doses chosen in this study were 1/10th of doses used intraperitoneally in neonatal rats that have been shown to be effective in evoking air-stepping behavior (Swann et al., 2016).

Results and Discussion

As can be seen in Figure 1A, the mean number of alternating hindlimb steps for each group receiving IT quipazine (dark bars) were slightly higher than for the group receiving IT saline (white bar). However, there were no statistically significant effects of drug condition on step frequency (F(3,28)=0.64, p=0.60, Figure 1A) or percentage of limb movements that were steps (F(3,28)=0.83, p=0.49, Figure 1B). Thus, the results from Experiment 1 indicate that low-thoracic IT administration of quipazine does not evoke significant differences in alternated hindlimb stepping compared to vehicle control.

Figure 1.

Figure 1.

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Alternating hindlimb stepping following IT quipazine administration, with saline as vehicle. A) Frequency of alternating hindlimb steps. B) Percentage of alternating hindlimb steps as a function of all hindlimb movements. Bars represent means; vertical lines depict SEM.

Following this unexpected finding, we piloted out numerous additional doses of quipazine using saline as vehicle control, which also were unsuccessful in yielding stepping behavior. Interestingly, it has been suggested that following spinal cord injury, neural circuits must be both primed for activity, and triggered into action by brain or sensory input (Fouad et al., 2010). Although our subjects did not have a spinal injury, we applied this reasoning to try to perhaps “unmask” effects of the IT drug administration, by applying a brief tail pinch in a few animals in follow-up pilot testing. Yet tail pinch only evoked a very brief response (Swann et al., 2017), but not much stepping like is typically seen following IP quipazine administration (Swann et al., 2016) (data not shown).

Our next step (Experiment 2) was to dissolve the quipazine in a different vehicle solution, to perhaps increase central nervous system accessibility to the drug. We also applied tail pinch to “trigger” spinal circuits, which in pilot testing appeared to reveal stepping better when applied with the new vehicle solution.

Experiment 2: Effect of Acute IT Quipazine Administration and Tail Pinch on Stepping Behavior, Using Tween 80 as Vehicle Control

Sakane and colleagues (1989) suggest that Tween 80 (Polysorbate 80) can disrupt the blood-brain barrier (BBB), and when used as a vehicle solution can allow drugs to more easily cross the BBB and enter the central nervous system. Therefore, in Experiment 2 we dissolved quipazine in Tween 80, before IT administration to newborn rats. Additionally, sensory stimulation, such as tail pinch, has been shown to induce stepping in neonatal rats and may have a synergistic, additive effect with quipazine to produce hindlimb stepping (Swann et al., 2017). Thus, the purpose of Experiment 2 was to address possible limitations of Experiment 1 by using a Tween 80-saline solution to dissolve quipazine and provide additional excitatory drive or a “trigger” to locomotor circuits through sacrocaudal afferent stimulation (i.e., tail pinch).

Experimental Design

A total of 24 P1 rats (12 males, 12 females) were used as subjects in Experiment 2. Subjects were assigned to one of three drug conditions: 0.3 mg/kg Quipazine, 1.0 mg/kg Quipazine, or vehicle control (93 % saline + 7% Tween 80 solution). As in Experiment 1, subjects received IT injection of 1 μL of solution at the low thoracic spinal level. Immediately following injection, subjects were suspended in the harness and prepared for behavioral testing. At 5-minutes post-injection, subjects received a tail pinch. The tail pinch was delivered using forceps to gently apply brief, mechanical pressure at the base of the tail (Lev-Tov et al., 2010; Norreel et al., 2003; Swann et al., 2017).

Pharmacology

Quipazine maleate was dissolved in a solution of 93% saline 7% Tween 80 and prepared in doses of 0.3 mg/kg, or 1.0 mg/kg. Given the null findings from Experiment 1, we did not replicate here the lowest dose of Quipazine (0.1 mg/kg) that was used in the previous experiment.

Results and Discussion

As can be seen in Figure 2, the groups receiving IT quipazine (gray bars) and tail pinch showed more alternating hindlimb steps (Figure 2A) and a higher percentage of steps (Figure 2B) than saline-treated animals (white bar). Although the difference was not significant for step frequency (F(2,21) = 1.09, p =0.35), it was significant for percentage of alternating steps (Figure 2B). A one-way ANOVA revealed a significant effect of drug condition (F(2,21) = 4.66, p < .05), such that subjects treated with 0.3 mg/kg of quipazine demonstrated a significantly higher percentage of alternating steps compared to vehicle controls (see Figure 2B). Although it is the case that these subjects were treated with a tail pinch in addition to IT quipazine, subjects in the vehicle control condition also were treated with a tail pinch. Therefore, under the conditions of providing sensory stimulation to the spinal cord, we were able to unveil an effect of IT quipazine on hindlimb stepping behavior in the newborn rat.

Figure 2.

Figure 2.

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Alternating hindlimb stepping following treatment with IT administration of quipazine with Tween 80 as vehicle, and in conjunction with tail pinch stimulation. A) Frequency of alternating hindlimb steps. B) Percentage of alternating hindlimb steps as a function of all hindlimb movements. Bars represent means; vertical lines depict SEM.

General Discussion

We found that IT administration of quipazine elicited hindlimb air-stepping in newborn rats. Specifically, IT quipazine, in conjunction with Tween 80 and sensory stimulation, induced a higher percentage of alternating steps in P1 rats compared to control subjects at the 0.3 mg/kg quipazine dose (Figure 2B). This finding suggests that quipazine acts directly on serotonin receptors within the spinal cord to activate locomotor circuitry, as has been suggested; and that effects of quipazine when administered intrathecally may be further enhanced by dissolving the drug in Tween 80 rather than just saline, for future studies.

Previous research has shown that the addition of Tween 80 to drug solutions significantly increases the absorption rate of the drug in adult rats (Kaneda et al., 1974; Zhang et al., 2003). Additionally, Sakane and colleagues (1989) found that Tween 80 disrupted the BBB, allowing increased drug uptake to the central nervous system. The BBB excludes a majority of both large and small molecule drugs, impacting the efficacy of drug delivery (Pardridge, 2005). Thus, with the addition of Tween 80 to the saline vehicle solution, quipazine was likely more readily absorbed and taken into the spinal cord via the BBB, contributing to the increased stepping response reported in the current study.

In addition to Tween 80, a brief tail pinch was applied in Experiment 2, to evoke stepping after IT quipazine treatment. In a previous report, we demonstrated that tail pinch evokes an immediate response in newborn rats, that lasts about 15s (Swann et al., 2017). The most comparable group in that study (to the present study) was sham-treated rats given IP 3.0 mg/kg quipazine, in which an average of about 15 hindlimb steps occurred in response to quipazine. Thus, we do not think it is likely that the tail pinch is responsible for producing the main effect of stepping following IT quipazine with Tween 80 that is reported here. Instead we suggest, as has been suggested for the injured spinal cord (Fouad et al., 2011), that sensory stimulation can help to trigger activity that is primed or organized by other means (i.e., pharmacologically). In the study reported here, it is not clear if that is a direct activation effect on spinal circuitry, or a release from inhibition. To further elucidate whether behavioral responses are affected by direct activation on spinal circuitry or release from inhibition, future studies should examine IT administration in spinal-transected animals. It is important to note that spinal cord transection physically disrupts the BBB and thus the response could be due to the injury rather than quipazine administration, and thus, it is important to understand the behavioral response in intact animals as a baseline. Nonetheless, it appears that the solution that was IT injected into the spinal cord clearly affected the behavioral response observed (see Figure 2B).

Although we tested and piloted different vehicle solutions and drug doses in examining the efficacy of IT quipazine administration inducing stepping behavior, more research can be done to better separate out and elucidate the effects of the solvent and tail pinch from one another. Yet at this point, given the weight of the evidence from spinal injured animals treated with IT or IP quipazine, and some mixed results on IT quipazine effectiveness, we suggest our results help to clarify the role of quipazine in eliciting spinal-mediated locomotor behavior. It may be the case that behavior or training differences across experiments are largely due to differences in testing protocols (i.e., drug doses, time since spinal injury, etc.). However, an important take away from the current study is that when quipazine is administered intrathecally to engage spinal locomotor circuitry, use of Tween 80 as the vehicle solution may yield more behavioral effects compared to saline.

Lastly, although IT administration of quipazine did not produce the same large number of hindlimb steps as typically seen with systematic administration of the drug (i.e., Strain & Brumley, 2014; Swann et al., 2016), this may be partly due to differences between a localized injection to the spinal cord and a systematic injection that can affect body systems more broadly. Serotonin receptors, including 5-HT2 receptors, are found throughout the body, including within the spinal cord, as well as the brain and gut (Liu & Jordan, 2005; Briejer, Mathis, & Schuurkes, 2003). For example, 5-HT2 receptors have been shown to mediate hypothalamic-pituitary-adrenocortical (HPA) axis activity, with receptor activation leading to increased serum corticosterone (Hemrick-Lueke & Evans, 2002). Additionally, Liu and Jordan (2005) reported that stimulation of the brain stem evoked locomotor-like activity involving 5-HT2A receptors. Therefore, other serotonin receptors throughout the body and brain, and not just localized within the spinal cord, are very likely to be involved in the response to systemic quipazine administration that may contribute to a more robust behavioral response following systemic drug administration in intact animals.

Acknowledgements

HEST and DDV were supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #P20GM103408. HEST was supported by an Idaho State University Undergraduate Research Grant, a Psi Chi International Honor Society Undergraduate Research Grant, and a Psi Chi Summer Research Grant.

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