Journal of Neuroscience Methods A new method to study sensory

Journal of Neuroscience Methods 182 (2009) 255–259

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Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth

Short communication

A new method to study sensory modulation of locomotor networks by activation of thermosensitive cutaneous afferents using a hindlimb attached spinal cord preparation Sravan Mandadi, Patrick J. Whelan ∗ Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada, T2N4N1

a r t i c l e

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Article history: Received 1 May 2009 Received in revised form 10 June 2009 Accepted 10 June 2009 Keywords: Spinal cord Mouse Afferents CPG TRP Sensory Nociceptors

a b s t r a c t The use of isolated in vitro spinal cord preparations to examine the underlying networks that control locomotion has become popular. It is also well known that afferent feedback can excite and modulate these networks. However, it is often difficult to selectively activate classes of afferents that subserve specific modalities using in vitro preparations. Here, we describe a technique where afferent receptors that detect temperature were selectively activated. To accomplish this we used an in vitro preparation of the mouse where the spinal cord was isolated (T5-cauda equina) with one hind limb left attached. We designed a special chamber allowing the hind paw to be placed in such a way that it remained attached to the spinal cord but received a separate supply of artificial cerebrospinal fluid (aCSF). This allowed us to alter the temperature of the hind limb compartment without affecting the temperature of the central compartment containing the spinal cord. We also demonstrate using this approach that agonists which activate receptors which detect noxious heat could be intradermally injected into the hind limb without it diffusing into the central compartment. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Sensory information from receptors in the hind limb is well known to control spinal circuits and a wealth of data has been generated using cat and rodent preparations (Pearson, 2004). Most of this work has focused on proprioceptors from muscle partly for historical and technical reasons. Part of the difficulty with activating cutaneous afferents is that they are heterogeneous and transduce sensation ranging from touch to pain (Lumpkin and Caterina, 2007). Consequently, relatively few tools have been available to selectively activate these classes of afferents. This has changed over the last 5 years with the cloning of ion channels which transduce temperature (Mandadi and Roufogalis, 2008). These are classes of transient receptor potential (TRP) ion channels which are present in skin receptors and can be activated by agonists or by changes in skin temperature. We focus here on two subclasses within the group of ThermoTRPs that have been well characterized for their role in somatosensation and nociception. These receptors have been found on both peripheral and central projections of C and A␦ cutaneous afferents (Caterina et al., 1997; McKemy et al., 2002; Bautista et al.,

∗ Corresponding author at: Department of Comparative Biology and Experimental Medicine, HS 2119, 3330 Hospital Drive NW, University of Calgary, Calgary Alberta, Canada. Tel.: +1 403 220 4210. E-mail address: [email protected] (P.J. Whelan). 0165-0270/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2009.06.011

2007). For example, transient receptor potential vanilloid 1 (TRPV1) is an ion channel that is activated by noxious heat (>43 ◦ C and, more specifically, by capsaicin) (Caterina et al., 1997). In contrast, transient receptor potential melastatin 8 (TRPM8) is an ion channel present primarily in a segregated group of primary afferents and is activated by cool temperatures (15–27 ◦ C) (McKemy et al., 2002). Therefore the prospect of examining the effects of modulating TRPs either by using agonists or by changing skin temperature and examining the effects on spinal motor networks is now possible. An important model for examining sensorimotor activation of spinal circuits is the isolated spinal cord preparation. This preparation has proven useful but lacks sensory input from the hind limb. To counter this issue, hind limb-spinal cord preparations have been developed with hind limb afferent input left intact. However, studies that have selectively activated receptors to examine locomotor function using this preparation have been limited. In this short communication, we outline the development of a technique to selectively activate cutaneous receptors in the mouse hind paw that transduce temperature sensation. We developed a hind limb attached to spinal cord preparation where we isolated the paw from the rest of the limb and the spinal cord. By doing so the paw could be superfused with different temperatures of artificial cerebrospinal fluid (aCSF) to selectively activate cutaneous afferents which in turn are known to activate specific classes of ThermoTRPs. In addition, intradermal skin injections of capsaicin activating cutaneous TRPV1 expressing afferents could be completed without any issues

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S. Mandadi, P.J. Whelan / Journal of Neuroscience Methods 182 (2009) 255–259

of diffusion into the aCSF superfusing the spinal cord. Part of the data used in this manuscript has been published (Mandadi et al., 2009).

use of animals. The remaining tissue was placed in a dissection chamber filled with oxygenated (95% O2 , 5% CO2 ) artificial cerebrospinal fluid (aCSF: concentrations in mM: 128 NaCl, 4 KCl, 1.5 CaCl2 , 1 MgSO4 , 0.5 Na2 HPO4 , 21 NaHCO3 , 30 d-glucose).

2. Methods 2.1. Chamber construction Experiments were performed on Swiss Webster mice (Charles River Laboratories, Senneville, Quebec, Canada). The animals were anesthetized by hypothermia, decapitated and eviscerated using procedures approved by the University of Calgary Animal Care Committee in conformation with international guidelines on the ethical

A recording chamber was custom manufactured with the following internal dimensions (5.7 cm (L) by 1.9 cm (W) by 1 cm (H) (Fig. 1). A dual superfusion system was constructed with separate inflows and outflows to allow the paw and spinal cord to be

Fig. 1. A schematic of the recording chamber used to record activity from a hind limb-attached spinal cord preparation (inset: photo of chamber). The chamber was constructed from plexiglass, and had two inflow and two outflow tubes. The chamber was split into two compartments by a plastic wall (constructed from standard laboratory weigh boats). The wall had a ‘U’ cut into it where the hind paw was placed. Vaseline was applied with an 18-gauge needle to completely separate the two compartments. Leaks between the two compartments were detected by using green food color which was applied to the compartment containing the hind paw. Thermistors were placed in each compartment to monitor the bath temperatures and were recorded along with neurogram data. The spinal cord compartment aCSF was maintained at 27 ◦ C while the hind paw compartment aCSF was variably adjusted between 17–45 ◦ C. A Hamilton syringe with a 33-gauge needle was mounted on a micromanipulator and used to inject drugs intradermally into the hind paw area.


separately superfused. Separate temperature controllers (Warner Instruments) allowed us to adjust the temperature of each compartment, and the temperature of each compartment was continuously monitored and recorded. A piece of thin plastic, (cut from standard lab weigh boats) was used to split the recording chamber (Fig. 1). A small ‘U’ shaped cut was made in the middle to accommodate the paw. The ‘U’ extended to about 1 cm above the base. This provided a stand on which to place the limb (Fig. 1). The film was then attached to the sides of the recording chamber using silicone. We applied a thin coat of sylgard to the base of the chamber to allow us to anchor the preparation with pins. 2.2. Tissue preparation

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pharmacological agents (N-methyl-d(l)-aspartic acid (NMA, 5 ␮M), dopamine (DA, 50 ␮M) and serotonin (5-HT, 10 ␮M)) to the spinal cord compartment (Whelan et al., 2000). Rhythmic alternating segmental L2 neurogram bursting along with alternating ipsilateral bursting between the L2 and L5 neurograms were taken to indicate fictive locomotion (Whelan et al., 2000). Data were analyzed using custom written programs (MatLab, MathWorks, Natick, Massachusetts) as well as commercially available programs (SigmaStat, Systat, San Jose, Ca; Oriana, Kovach Computing Services, Anglesey, UK). We made use of cross-correlograms to examine the strength of coupling between neurograms and autocorrelograms to calculate the rhythm frequency. Full details of these analyses have been previously published (Madriaga et al., 2004; Pearson et al., 2003).

A dorsal and ventral laminectomy exposed the spinal cord sparing as much of the cauda equina as possible. The cord was transected at T5 with the spinal cord attached to a single hind limb by the lumbar and sacral dorsal roots. The remaining thoracic dorsal and ventral roots were cut. The hind limb-spinal cord preparation was then transferred to a recording chamber filled with oxygenated aCSF. The paw was placed into the ‘U’ hole in the plastic and sealed using Vaseline jelly. The Vaseline was applied using a 10 ml syringe with an 18-gauge needle (tip was filed off). The aCSF superfusing the spinal cord portion was equilibrated at room temperature before being heated to approximately 27 ◦ C. The aCSF superfusing the compartment containing the hind paw was heated to approximately 32 ◦ C. To ensure that no leaks occurred between the compartments green food color was added to the hind paw compartment. If any green color was observed in the spinal cord compartment, then the experiment was aborted. A noxious heat ramp (60 s) was applied to the compartment containing the hind paw using preheated aCSF. We then maintained the hind paw compartment at 45 ◦ C. The temperature was then returned to 32 ◦ C. In separate experiments a non-noxious cold ramp was delivered to the hind paw compartment using pre-cooled aCSF. This enabled us to generate a cooling ramp which reached approximately 17 ◦ C within 60 s and was then maintained at 17 ◦ C using the temperature controller. During application of all temperature ramps to the hind paw, the preparation was allowed to equilibrate for 10 min at different temperatures before recordings were made for an additional 10 min. The spinal cord compartment temperature was changed by a small amount following the temperature ramps (0.4–1 ◦ C). In separate experiments we found that these small temperature fluctuations were not by themselves sufficient to change rhythm parameters (data not shown). Intradermal capsaicin injections to the hind paw were made using a Hamilton syringe fitted with 33-gauge needle and mounted on a micromanipulator. Control experiments were carried out to eliminate the possibility of effects on the rhythm due to the vehicle (dimethyl sulfoxide (DMSO)) or to the injection procedure itself. In order to account for mechanical stimulation of the hind paw by the needle, the preparation was allowed to equilibrate for 10 min after the needle was introduced. Then, capsaicin (1 ␮M; 1 ␮l) was injected into the dorsal skin of the hind paw to selectively activate TRPV1. For all intradermal injection experiments, the spinal cord and the hind limb portion of the bath were maintained at approximately 27 ◦ C and 32 ◦ C, respectively.

3. Results

2.3. Electrophysiological recordings

Control Hindpaw heated Heat withdrawn Control Capsacin 1 ␮M Control Hindpaw cooled Cooling withdrawn

Our initial tests of the chamber showed that we could maintain separate temperatures of the compartment containing the spinal cord and part of the leg, and the compartment containing the paw. In terms of placing the hind limb and sealing the two compartments, we found the best approach was to first place a small amount of Vaseline using an 18-gauge needle in the base of the ‘U’ and then lay the hind limb on top. The Vaseline was then applied in a layered fashion to completely seal the two compartments. Overall, this approach resulted in very few failures. We first tested the possible modulation of locomotor rhythms by activation of thermoreceptors in the hind paw. All ventral roots were removed to block movement of the hind paw. Otherwise, we would not have been able to maintain the integrity of the Vaseline dams. The lumbosacral dorsal roots from the hind limb were not cut. Rhythmic motor activity was evoked by bath application of NMA (5 ␮M), 5-HT (10 ␮M), and dopamine (50 ␮M) to the spinal cord compartment (Fig. 2A–C) (Jiang et al., 1999; Whelan et al., 2000). Once a stable rhythm was achieved, we systematically changed the temperature of the hind limb compartment aCSF. We first investigated recruitment of cutaneous nociceptive afferents by application of noxious heat (approximately 43 ◦ C) to the hind paw. Noxious heat resulted in a significant increase in the frequency of the rhythm as reflected by a decrease in the cycle period (Fig. 2A and Table 1, n = 4). Given that multiple TRP channels (TRPV1, TRPV3 & TRPV4) encode heat within the temperature range of approximately 32 ◦ C to 45 ◦ C, we next examined whether activation of the noxious heat (>43 ◦ C) detector TRPV1 by intradermal injection of capsaicin, which selectively activates TRPV1, would have a similar effect on locomotor output. Within 5 min of the capsaicin injection, there was significant increase in the frequency of the rhythm as reflected by a decrease in the cycle period (Fig. 2B and Table 1, n = 4). Cooling the hind paw using temperatures ranging from 32 ◦ C to 17 ◦ C likely activates TRPM8 channels (Bautista et al., 2007). To specifically address recruitment of TRPM8 expressing cutaneous afferents which are known to encode non-noxious cool temperatures, we separately Table 1 Mean cycle period of the fictive locomotor rhythm was significantly altered by noxious heat, capsaicin and non-noxious cold. Treatment groups

Mean cycle period ± SEM (s) L2-L2

Neurograms were recorded with suction electrodes into which typically the left and right lumbar 2 (L2) and 5 (L5) ventral roots were drawn. The neurograms were amplified (100–20,000 times), filtered (0.1–1 kHz or DC–1 kHz) and digitized (Axon Digidata 1322A, Molecular Devices, Sunnyvale, CA) for future analysis. Fictive locomotion was evoked by bath applying a combination of

a

3.79 3.06 3.94 3.50 2.92 3.09 4.29 3.11

± ± ± ± ± ± ± ±

L2-L5 0.20 0.01a 0.29 0.04 0.18a 0.05 0.04a 0.03

p < 0.05, ANOVA by repeated measures; N = 4.

3.78 3.05 4.02 3.49 2.92 3.19 4.18 3.18

± ± ± ± ± ± ± ±

0.23 0.10a 0.35 0.04 0.17a 0.09 0.07a 0.09

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Fig. 2. Changing the temperature of the aCSF superfusing the hind paw alters the frequency of the locomotor-like rhythm. The rhythm was evoked by bath application of NMA (5 ␮M), 5-HT (10 ␮M), and dopamine (50 ␮M). Neurograms were obtained from the L2 and L5 ventral roots of the spinal cord. (A) Recordings from the same animal showing that when the temperature of the hind paw was increased from 32 ◦ C (left panel) to 45 ◦ C (middle panel), the rhythm frequency increased. After the noxious heat was withdrawn the rhythm returned to control frequencies (right panel). (B) At a skin temperature of 32 ◦ C, intradermal injection of capsaicin to the dorsal skin of hind paw, which activates TRPV1, caused the rhythm to speed up (right panel). (C) When the skin temperature was cooled from 32 ◦ C (left panel) to 17 ◦ C (middle panel), the rhythm slowed down. The rhythm returned to control conditions when the cooling was withdrawn (right panel). In all experiments, the preparations were allowed to equilibrate for 10 min before recordings were obtained.

applied cool temperatures of 17 ◦ C to the hind paw. The rhythm was affected shortly after cooling and reached a steady state 10 min after the change in temperature. Overall, there was a significant slowing of the rhythm reflected by an increase in the cycle period (Fig. 2C and Table 1, n = 4). Following re-warming of the hind paw to control temperatures (approximately 32 ◦ C), the rhythm frequency returned to control values (Fig. 2C and Table 1). Noxious heat or non-noxious cooling did not change the phase (Modified Rayleigh’s, p < 0.01; Watson-Williams, p > 0.5; data not shown). Our results suggest that our technique can be used to identify cutaneous afferents that selectively encode noxious heat or non-noxious cool temperatures and modulate fictive locomotor rhythms. 4. Discussion In this work we describe a method to isolate the hind paw using a modified dual-perfusion system. Our goal was to create a method to discretely activate cutaneous afferents using in vitro spinal cord preparations. To validate this method we present data showing that changes in the temperature of the hind paw compartment aCSF can activate cutaneous afferents that encode temperature and produce changes in the locomotor network. We found that increased aCSF temperatures could increase the frequency of the motor rhythm; an effect that could be mediated by a number of TRP channels including TRPV3 and TRPV4 which encode warm temperatures

(Mandadi and Roufogalis, 2008), and TRPV1 which encodes noxious heat (Caterina et al., 1997). Likewise, similar increases in rhythm frequency were observed when capsaicin was injected, which is known to activate TRPV1 (Caterina et al., 1997). Our method is not the only reported way to activate thermoreceptors. A rat hind limb-tail-spinal cord preparation was used to activate sacrocaudal nociceptors by radiant heat or a pinch to the tail (Blivis et al., 2007). While this is a useful method to activate noxious heat receptors, our method has the advantage of being able to activate thermoreceptors across the spectrum from noxious heat to cold. An additional advantage is that the temperature of the two chambers can be independently modified and are recorded onto separate channels along with the electrophysiological traces. This allows the experimenter to have a permanent record of the temperature during the entire experiment. Every new method has its limitations. The main limitation of our approach is that it is necessary to cut the ventral roots. Cutting the ventral roots immobilizes the hind limb, so that the hind paw does not move once a seal is formed between the two chambers. This means that it is not possible using the described method to record electromyograms (EMGs) from the hind limb muscles. Recording EMGs from hind limb muscles provides more precision when examining changes in the pattern of locomotion (Cowley and Schmidt, 1994). If this type of data are required, then it should be possible to adapt the methods here. For example, one could record electroneu-


rograms from the lateral gastrocnemius and soleus (LGS) or medial gastrocnemius (MG) nerve (Whelan et al., 2000) while paralyzing the hind limb using gallamine triethiodide (flaxedil). That said, we believe that our preparation is an attractive method to examine selective recruitment of temperature sensitive afferents and their effects on motor circuits. Also the mouse provides the added advantage of providing genetic models to delineate subsets of muscle and cutaneous afferents which encode an array of sensory modalities. Acknowledgements We would like to thank Michelle Tran for her excellent technical assistance. We greatly appreciate ongoing support from the Alberta Heritage Foundation for Medical Research, the Canadian Institutes of Health Research, and the University of Calgary. Dr. Sravan Mandadi was supported by a fellowship from the Alberta Heritage Foundation for Medical Research. References Bautista DM, Siemens J, Glazer JM, Tsuruda PR, Basbaum AI, Stucky CL, et al. The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 2007;448:204–8.

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