Nociceptive responses of midbrain dopaminergic neurones are modulated by the superior colliculus in the rat

Neuroscience 139 (2006) 1479 –1493

NOCICEPTIVE RESPONSES OF MIDBRAIN DOPAMINERGIC NEURONES ARE MODULATED BY THE SUPERIOR COLLICULUS IN THE RAT V. COIZET,1* E. J. DOMMETT,1 P. REDGRAVE AND P. G. OVERTON

Key words: electrophysiology, dopamine, pain, nociception, sensory regulation.

Department of Psychology, University of Sheffield, Western Bank, Sheffield S10 2TP, UK

Dopamine-mediated transmission has been implicated in a number of human clinical disorders, including Parkinson’s disease and schizophrenia, as well as in a wide range of normal brain functions, including the processes that underlie associative learning. Typically, dopaminergic (DA) neurones exhibit a stereotyped, short-latency (⬍100 ms), short duration (⬃100 ms) population response to unpredicted stimuli in various modalities that are salient by virtue of their novelty, intensity or reward value (Freeman and Bunney, 1987; Horvitz et al., 1997; Schultz, 1998). While much is known about many aspects of the ascending DA systems, surprisingly little is known about the sensory inputs that modulate their phasic activity. In the case of vision, the latencies of the phasic DA responses led us to investigate the possibility that a subcortical visual structure, the superior colliculus (SC), rather than a cortical relay, might be the critical source of afferent visual input (Comoli et al., 2003; Dommett et al., 2005). Visual response latencies of SC neurones (40 – 60 ms; Wurtz and Albano, 1980; Munoz and Guitton, 1986; Jay and Sparks, 1987; Peck, 1990; Stein and Meredith, 1993), are consistently shorter than those of DA neurones (70 –100 ms; Schultz, 1998; Morris et al., 2004; Takikawa et al., 2004), while responses in cortical regions responsible for feature detection and object recognition are approximately the same or longer (80 –130 ms) (Thorpe and Fabre-Thorpe, 2001; Rousselet et al., 2004). Our investigations have led to the discovery of a significant, previously unreported direct projection from the SC to the substantia nigra (SN) pars compacta and ventral tegmental area (VTA) in the rat, which innervates both DA and non-DA neurones in these regions (Comoli et al., 2003). Associated electrophysiological experiments revealed that SC is a critical relay for short-latency visual evoked potentials recorded locally in the SN pars compacta (Comoli et al., 2003). Furthermore, we have recently reported (Dommett et al., 2005) that local disinhibition of the deep layers of the SC of anesthetized rats is both sufficient and necessary for light stimuli to evoke shortlatency phasic activation of DA neurones. Comparable disinhibition of the visual cortex had no effect, leaving midbrain DA neurones insensitive to the light stimulus. Together, these data suggest that in the rat, the SC is the primary, if not the exclusive, source of short-latency visual

Abstract—Midbrain dopaminergic neurones exhibit a shortlatency phasic response to unexpected, biologically salient stimuli. In the rat, the superior colliculus is critical for relaying short-latency visual information to dopaminergic neurones. Since both collicular and dopaminergic neurones are also responsive to noxious stimuli, we examined whether the superior colliculus plays a more general role in the transmission of short-latency sensory information to the ventral midbrain. We therefore tested whether the superior colliculus is a critical relay for nociceptive input to midbrain dopaminergic neurones. Simultaneous recordings were made from collicular and dopaminergic neurones in the anesthetized rat, during the application of noxious stimuli (footshock). Most collicular neurones exhibited a short-latency, short duration excitation to footshock. The majority of dopaminergic neurones (92/110; 84%) also showed a short-latency phasic response to the stimulus. Of these, 79/92 (86%) responded with an initial inhibition and the remaining 14/92 (14%) responded with an excitation. Response latencies of dopaminergic neurones were reliably longer than those of collicular neurones. Tonic suppression of collicular activity by an intracollicular injection of the local anesthetic lidocaine reduced the latency, increased the duration but reduced the magnitude of the phasic inhibitory dopaminergic response. These changes were accompanied by a decrease in the baseline firing rate of dopaminergic neurones. Activation of the superior colliculus by the local injections of the GABAA antagonist bicuculline also reduced the latency of inhibitory nociceptive responses of dopaminergic neurones, which was accompanied by an increased in baseline dopaminergic firing. Aspiration of the ipsilateral superior colliculus failed to alter the nociceptive response characteristics of dopaminergic neurones although fewer nociceptive neurones were encountered after the lesions. Together these results suggest that the superior colliculus can modulate both the baseline activity of dopaminergic neurones and their phasic responses to noxious events. However, the superior colliculus is unlikely to be the primary source of nociceptive sensory input to the ventral midbrain. © 2006 Published by Elsevier Ltd on behalf of IBRO. 1

These authors contributed equally to this work. *Corresponding author. Tel: ⫹44-0-114-222-6598; fax: ⫹44-0-114276-6515. E-mail address: [email protected] (V. Coizet). Abbreviations: ANOVA, analysis of variance; BSA, bovine serum albumin; CED, Cambridge Electronic Design; DA, dopaminergic; DAB, diaminobenzidine; FLI, Fos-like immunoreactivity; NGS, normal goat serum; NHS, normal horse serum; PAG, periaqueductal gray; PB, phosphate buffer; PBS, phosphate-buffered saline; PSTH, peri-stimulus time interval histogram; SC, superior colliculus; SN, substantia nigra; TH, tyrosine hydroxylase; TX, Triton X-100; VTA, ventral tegmental area. 0306-4522/06$30.00⫹0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2006.01.030

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input to midbrain DA neurones, possibly mediated via direct tectonigral projections (Comoli et al., 2003). However, the SC is a multi-sensory structure containing neurones which respond to visual, auditory and somatosensory stimulation (Drager and Hubel, 1975; Chalupa and Rhoades, 1977; Stein and Meredith, 1993; Wallace et al., 1996). The present investigation was therefore designed to test whether the SC is the primary source of sensory input to DA neurones for a modality other than vision. Because of the hypothesized central role of DA in signaling errors in predicted reward (Schultz, 1998), we chose to investigate whether the SC plays a critical role in signaling the occurrence of noxious events to DA neurones. The SC appears to play an important role in nociception, at least in the rodent. Hence, the intermediate and deep layers of the rat and hamster colliculus receive direct input from the nociceptive layers of the spinal cord (Rhoades, 1981; Almeida et al., 2004). These SC layers contain neurones that resemble spinal wide dynamic range and nociceptive specific (high threshold) cells, which respond with a short-latency tonic increase in activity to noxious mechanical and/or thermal stimulation (Stein and Dixon, 1979; McHaffie et al., 1989; Redgrave et al., 1996a). Many nociceptive SC neurones in the rat send efferent projections via the predorsal bundle to the contralateral brainstem (Redgrave et al., 1996a). Disruption of this projection prevents the effective execution of approach movements to attack or ameliorate a source of discomfort (Redgrave et al., 1996b; Wang and Redgrave, 1997). DA neurones also respond to noxious stimuli in a wide range of species, including the rat (Tsai et al., 1980; Maeda and Mogenson, 1982; Kiyatkin and Zhukov, 1988; Mantz et al., 1989; Gao et al., 1990; Ungless et al., 2004), rabbit (Guarraci and Kapp, 1999) and monkey (Schultz and Romo, 1987; Mirenowicz and Schultz, 1996). In the rat, noxious stimuli produce a short-latency change in discharge frequency. Although the nociceptive responses of DA neurones identified by electrophysiological criteria alone have been reported to be either an increase, or more commonly, a decrease in firing (Tsai et al., 1980; Maeda and Mogenson, 1982; Kiyatkin and Zhukov, 1988; Mantz et al., 1989; Gao et al., 1990; Ungless et al., 2004), a recent study by Ungless et al. (2004) suggested that the phasic nociceptive response of neurochemically identified DA neurones is uniformly inhibitory. The purpose of the present study was to ascertain whether the SC is the critical source of short latency nociceptive input to DA neurones. Our study contained three components: (i) A single noxious electrical footshock was used to investigate the timing relationship between SC and DA nociceptive responses. If the SC is a critical relay for nociceptive information its latencies would be expected to be shorter than those of DA neurones. (ii) We examined the effects of chemical manipulation of the SC on the phasic responses of DA neurones to noxious stimuli. Again, if the SC is a critical relay, these manipulations would be expected to have a dramatic effect on the DA nociceptive response. (iii) We tested the effects of remov-

ing the ipsilateral SC on the capacity of DA neurones to respond to noxious stimuli.

EXPERIMENTAL PROCEDURES Subjects Data were obtained from 44 female Hooded Lister rats (bred in our laboratory), weighing 220 –300 g at the time of study. All aspects of this study were performed with Home Office approval under section 5(4) of the Animals (Scientific Procedures) Act of 1986, and experimental protocols received prior approval by our institutional ethics committee. Every effort was made to minimize suffering and reduce the number of animals used.

Surgical preparation Animals were anesthetized with an i.p. injection of urethane (ethyl carbonate; 1.25 g/kg as a 25% aqueous solution) and mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) with the skull level. Body temperature was maintained at 37 °C with a thermostatically controlled heating blanket. Two stainless steel electrodes (E363-1, Plastics One, Roanoke, VA, USA) were inserted subcutaneously into the left hindpaw, one under the plantar surface and the other into the medial aspect of the ankle. Craniotomies were then performed to allow access to the SC and SN pars compacta.

Recording and injection procedure Extracellular single unit recordings were made from DA neurones located contralaterally to the stimulated hindpaw using glass microelectrodes (Narashige Laboratory Instruments Ltd., Tokyo, Japan) with tip diameters of approximately 1 ␮m (impedances 5–20 M⍀, measured at 135 Hz in 0.9% NaCl). Electrodes were filled with 0.5 M saline and 2% Pontamine Sky Blue (BDH Chemicals Ltd., Poole, UK). In each experiment, the electrode was lowered to a position dorsal to the SN pars compacta (AP, 4.8 – 6.04 mm caudal to bregma, DV, 6.5–7.5 mm ventral to the brain surface). An angled (35°) contralateral approach (beginning 2.6 –3.0 mm lateral to midline on the opposite side of the brain) was used to avoid collision with a second electrode inserted vertically into the SC (see below). Spike related potentials were amplified, band-pass filtered (300 Hz–10 kHz), digitized at 10 kHz and recorded directly onto computer disc using a 1401 plus data acquisition system (Cambridge Electronic Design [CED] Systems, Cambridge, UK) running CED data capture software (Spike 2). In animals undergoing chemical manipulation of the SC (N⫽22), a 30 gauge metal injector needle, filled with either bicuculline methiodide (100 ng/␮l in 0.9% saline; Sigma-Aldrich, Poole, UK) or lidocaine (40 ␮g/␮l in distilled water; Sigma-Aldrich), was coupled to a Parylene-C-coated tungsten electrode (2 M⍀; A-M Systems Inc., Carlsborg, WA, USA). The assembly was introduced vertically into the SC (AP, 6.3–7.3 mm caudal to bregma, ML, 1.0 –2.0 mm lateral to midline). Lateral separation between the electrode and the injector was 0.2– 0.5 mm. Intracollicular injections (0.5 ␮l; 0.5 ␮l/min) were made with a 10 ␮l Hamilton syringe (Scientific Laboratory Supplies Ltd., Nottingham, UK) mounted on an infusion pump, connected to the needle by a length of plastic tubing. Electrophysiological responses were determined while the electrode/injector assembly was lowered into the SC in the presence of a whole field light flash (0.5 Hz, 10 ms duration) from a green LED (60 lux 570 nm) positioned 5 mm in front of the contralateral eye. Using the characteristic vigorous visual response of the superficial layers of the SC as a positional cue, the electrode was lowered into the intermediate layers (3.0 –5.0 mm ventral to the brain surface). In accordance with the somatosensory representation of the body in the SC (McHaffie et al., 1989),

V. Coizet et al. / Neuroscience 139 (2006) 1479 –1493 the electrode/injector was aimed caudally into the foot area. In some of the earlier experiments (N⫽4), a single uncoupled tungsten electrode, or glass micropipette was used to record SC single unit activity in the absence of chemical modulation. An additional group of animals (N⫽6) received aspiration lesions of the SC (and overlying cortex) performed with a blunted 20 g hypodermic needle connected to a vacuum pump. This procedure was designed to produce a complete unilateral ablation of the SC ipsilateral to recorded DA neurones.

Sensory stimulation procedures Once the SC electrode/injector had been positioned, the DA electrode was lowered until a putative DA neurone was identified using the standard electrophysiological criteria of Grace and Bunney (1983): long action potential duration (⬎2.0 ms), low firing rate (⬍10 Hz) and a firing pattern that consisted of irregular single spikes or bursts; and also, the recently suggested criterion of the first phase of the action potential exceeding 1.1 ms (Ungless et al., 2004). Once encountered, the activity of the presumed DA-cell, and multiunit activity in the SC were recorded during the successive application of three different somatosensory stimuli to the hindpaw: (i) non-noxious somatosensory stimulation: stroking the plantar surface of the foot with a paintbrush (⬃0.5 s stroke along the long axis of the foot, at approximately 0.5 Hz); (ii) noxious mechanical pinch: the pinch was applied between the dorsum pedis and plantar surface with toothed forceps (⬃0.5–1.0 s pinch at approximately 0.5 Hz); and (iii) noxious footshock (single pulses at 0.5 Hz, 2 ms duration, 3.0 –5.0 mA). These electrical stimulation parameters are approximately three times the threshold for activating C-fiber (Chang and Shyu, 2001; Matthews and Dickenson, 2001; Carpenter et al., 2003) and have been shown to produce reliable A␦ and C-fiber responses in the anesthetized rat spinal cord (Urch et al., 2003). After determining the electrophysiological responses of the SC and SN pars compacta to 30 stroke stimuli, 30 pinch stimuli and 60 footshocks in the absence of any chemical manipulations, an injection of either lidocaine or bicuculline was made into the SC. Typically these treatments evoked a change in local SC multiunit activity within 60 –120 s of the injection. Noxious footshock was applied throughout the period of the SC injection and continued until either the effects of the drug wore off in the SC, or the DA neurone was lost. After a trial was completed, further DA neurones were sought and the testing procedures repeated. The nociceptive responses of between one and five DA neurones were tested in a single subject.

Aspiration lesions In animals in which the SC was to be lesioned, the responses of a single DA neurone were recorded to the range of somatosensory stimuli (see above). The ipsilateral SC and overlying cortical tissue was then removed by aspiration. Further DA neurones were then sought and their responses to the range of somatosensory stimulation determined. Again, up to five DA neurones were recorded from each animal post-aspiration.

Histology In each case the final recording site of the DA electrode was marked with Pontamine Sky Blue by passing a constant cathodal current of 27.5 ␮A (constant current source: Fintronics Inc., Orange, CA, USA) for a period of 30 min. Animals were then killed with an overdose of barbiturate and perfused transcardially with 400 ml of warmed saline (40 °C), followed by 400 ml of paraformaldehyde in phosphate buffer (PB, pH 7.4). Brains were removed and post-fixed overnight in 4% paraformaldehyde at 4 °C, before being transferred into sucrose for 36 h. Serial coronal (30 ␮m) sections were cut in a cryostat. One series of sections

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was mounted on slides and processed with a Nissl stain (Cresyl Violet), while a second series was collected in 0.1 M PB and processed for tyrosine hydroxylase (TH), and in some cases, c-fos immunohistochemistry as follows: First, sections were blocked in 0.1 M PB–Triton X-100 (TX) 0.3% with 2.5% bovine serum albumin (BSA) and 5% normal horse serum (NHS) for 2 h. They were then incubated overnight with the primary mouse monoclonal antibody diluted 1:3000 (Chemicon, Hampshire, UK) in 0.1 M PB-TX 0.3% with 1% BSA and 2% NHS. The following day, sections were washed in 0.1 M PB and incubated with the secondary antibody, biotinylated antimouse made in horse (in a dilution of 1:1000 in 0.1 M PB-TX 0.3% with 2% NHS) for 2 h. Following further washes in 0.1 M PB, the sections were exposed to the elite Vectastain ABC reagent (Vector Laboratories, Burlingame, CA, USA) diluted 1:100 in PB-TX 0.3%, again for 2 h. Again following washes in 0.1 M PB, immuno-reactivity was revealed by exposure to VIP (Vector Laboratories) for 3 min which produced a purple reaction product. Sections were then mounted onto gelled slides, dehydrated through alcohols and cleared in xylene before being coverslipped with DPX. Recording sites were reconstructed onto sections taken from the atlas of Paxinos and Watson (1997). In animals with SC aspirations, lesions were reconstructed from photomicrographs taken using an RT Color Spot camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA) and Nikon Eclipse E800 microscope (Nikon Instruments, Kingston-uponThames, UK). In cases where bicuculline had been injected into the SC, sections were processed for the functional activity marker c-fos (Herdegen and Leah, 1998) prior to TH processing. Animals were left for 2 h after the bicuculline injection to permit time for expression of the Fos-protein before final perfusion (Herdegen and Leah, 1998). To reveal fos-like immunoreactivity (FLI), free-floating sections were first washed in 0.1 M PB then incubated in a blocking solution containing 0.1 M PB-TX 0.3% with 2.5% BSA and 5% normal goat serum (NGS) for 2 h. Sections were then incubated overnight with the primary polyclonal rabbit antibody to Fos protein (Merck Biosciences, Nottingham, UK) diluted 1:20,000 in phosphate-buffered saline (PBS)–TX 0.25% with 1% BSA and 2% NGS. The following day, sections were washed in PB and then incubated with the secondary antibody, biotinylated anti-rabbit IgG made in goat (1:600, Vector Laboratories) in PB–TXS 0.25% with 2% NGS for 2 h. After washes in PB, the sections were exposed to ABC (Vector Laboratories; 1:50 in PBS–TX 0.5%) for 2 h. Following further washes in PB, immunoreactivity was revealed by exposing the sections for 6 –10 min to diaminobenzidine (DAB) with hydrogen peroxide in 0.1 M PB. Immunoreactivity was enhanced with 1% ammonium nickel sulfate and 1% cobaltous chloride to reveal a black reaction product. After processing for FLI, additional TH immunohistochemistry was performed as described above to produce a contrasting purple stain.

Electrical stimulation and spinal cord c-fos expression Expression of the neural activity marker c-fos was also used to confirm the presence of activity in nociceptive layers of the spinal cord of four rats in response to the footshock used in the electrophysiological experiments. Animals were anesthetized with urethane and electrodes inserted into the left hindpaw (see above). Two animals received 1 h of electrical stimulation (see above). After a further hour they were perfused transcardially with 400 ml of warmed saline (40 °C), followed by 400 ml of paraformaldehyde in PB (pH 7.4), and the spinal cord was removed. In the remaining two (control) animals, the electrodes were positioned into the foot but no stimulation was delivered. Spinal cords were post-fixed overnight in 4% paraformaldehyde at 4 °C, then transferred into sucrose for 36 h. The lumbar region of the spinal cord was embedded in OCT (Sakura, Zoeterwoude, The Netherlands), frozen and cut in the coronal

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plane at 50 ␮m. Free floating sections were washed with 0.1 M PBS to remove traces of the OCT. They were then incubated for 2 h in a blocking solution of 0.1 M PB containing 0.3% TX with 2.5% BSA and 5% NGS. Sections were incubated overnight in the primary antibody, anti-c-fos (made in rabbit PC 38, Merck Biosciences) diluted 1:20,000 in 0.1 M PBS containing 0.25% TX with 1% BSA and 2% NGS. The next day, the sections were washed with 0.1 M PB and incubated for 2 h with the secondary antibody, biotinylated anti-rabbit IgG (made in goat; Vector Laboratories) in the dilution 1:600 in 0.1 M PB containing 0.25% TX and 2% NGS. After further washing the sections were exposed to ABC (Vector Laboratories) made in 0.1 M PB containing 0.5% TX. Immunoreactivity was revealed by exposing the tissue for 10 –15 min to DAB with hydrogen peroxide in 0.1 M PB, enhanced with 1% ammonium nickel sulfate and 1% cobaltous chloride. Finally, sections were mounted onto gelled slides, dehydrated with alcohol, cleared in xylene and coverslipped with DPX.

Data analysis Characteristics of the spike waveforms generated by DA neurones were determined during the experiment by averaging 200 –300 spikes. In the case of the SC multiunit data, data files were high pass filtered to reduce the electrical-stimulus artifact. Activity of both DA neurones, and SC single units, was separated from noise and stimulus artifact using template matching routines (Spike 2; CED). Peri-stimulus time interval histograms (PSTHs) were constructed based on DA (and SC) single unit and SC multiunit data (bin width 20 ms and 1 ms respectively). PSTHs were used to determine the latency and duration of stimulus-evoked responses. Post-stimulation deviations in activity that exceeded 1.96 S.D. of the pre-stimulation baseline (measured over 100 –500 ms before the stimulus) for DA and SC single units, and 3.00 S.D. for SC multiunit activity, were recorded as the point of response onset. Response offset was recorded when post-stimulation activity returned with these threshold values. Evoked responses in accord with these criteria were classified as excitatory, inhibitory or non-responsive. Effects of chemical modulation of the SC on the activity patterns of DA neurones was analyzed for the period during which local SC activity was affected by the drug. This period was defined when post-injection activity in the SC (200 ms post-stimulation) deviated by ⬎1.96 S.D. of baseline values determined during the 60 pre-drug stimulations. Quantitative differences in response characteristics were assessed with t-tests, analyses of variance (ANOVAs) and correlation analysis (accepted significance level P⬍0.05, two tailed). For animals undergoing SC aspiration, the relative frequencies of nociceptive DA neurones and non-responsive DA neurones were tested with chi square (␹2) analysis against the frequencies encountered in the entire population of intact animals.

RESULTS Nociceptive activity in the spinal cord Noxious footshock (0.5 Hz) for 1 h induced an expression of FLI that was concentrated in the medial part of the ipsilateral superficial layers of the cord, especially layers I and II, although layers III–V also contained some FLI (Fig. 1A). This pattern was particularly evident at L3-5. In control animals, where electrodes were implanted but no stimulation applied, substantially lower levels of FLI were observed (Fig. 1B). These results confirm that the footshock used in the present study was activating nociceptive elements in the lumbar spinal cord (Besson, 1987; Almeida

et al., 2004) consistent with known somatotopic representations of the hind foot; i.e. primary afferents from the foot terminate medially (Swett and Woolf, 1985). Effects of noxious stimulation on collicular activity Non-noxious somatosensory stimuli (brushstrokes) failed to evoke any response in isolated single units (N⫽11) in the caudal SC (Fig. 2A). However, most (10/ 11) of the same units showed an excitatory response to both pinch (Fig. 2B), and electrical stimulation (Fig. 2C). Nociceptive neurones in the SC were located primarily in the intermediate and deep layers (Fig. 2D). The phasic excitatory responses to footshock in isolated SC units had very short onset latencies (9.9⫾7.2 ms) and brief duration (12.0⫾14.2 ms). Multiunit responses (N⫽18) to somatosensory stimulation were also obtained from the intermediate and deep layers (Fig. 2E). Again, no phasic responses were elicited by non-noxious somatosensory stimulation, but both pinch (data not shown) and footshock (Fig. 2F) elicited phasic excitatory responses. Multiunit responses to the footshock had a short onset latency (6.5⫾0.2 ms) and duration (16.0⫾1.6 ms), similar to single unit responses. Effects of somatosensory stimulation on the activity of DA neurones All putative DA neurones sampled from intact animals in the present study (N⫽110; e.g. Fig. 3A) had firing rates (mean 4.3⫾0.2 Hz) and action potential waveform durations (total duration, mean⫾1 S.E.M.⫽5.9⫾0.1 ms) which met the criteria of Grace and Bunney (1983). Almost all (106/110 96%) also met the waveform duration criterion of Ungless et al. (2004) (initial duration⫽1.5⫾0.03 ms). Furthermore, in the majority of cases neurones were located in the TH-immunoreactive region of the ventral midbrain corresponding to the SN pars compacta (Fig. 3B); occasional neurones were found in the subjacent SN pars reticulata, which is known to contain some ventrally displaced DA neurones (Richards et al., 1997). Nigral DA neurones also showed no phasic response to non-noxious somatosensory stimulation (Fig. 3C top). However, a large majority, 92/110 (84%), showed a phasic response to noxious pinch. Of these, 79 (86%) responded with an initial inhibition and the remaining 13 (14%) responded with an initial excitation (e.g. Fig. 3C middle left and right). These 92 neurones also showed a response of the same sign (inhibition or excitation) to footshock (e.g. Fig. 3C bottom). The latency of the inhibitory responses to footshock was significantly shorter than their excitatory counterparts (63.5⫾3.2 ms vs 136.9⫾14.3 ms respectively; t⫽4.7, df⫽90, P⬍0.001), although the duration of inhibitory and excitatory responses was not reliably different (172.1⫾14.8 ms vs 114.6⫾9.7 ms respectively; t⫽1.5, df⫽90, P⬎0.05). Multiunit nociceptive responses in the SC had significantly shorter onset latencies than both inhibitory (t⫽8.4, df⫽95, P⬍0.001) and excitatory DA responses (t⫽5.0, df⫽95, P⬍0.001).

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Fig. 1. Coronal sections of the lumbar region of the spinal cord processed for c-fos expression. (A) Sections from segments of the lumbar region of the spinal cord in an animal subjected to 1 h unilateral noxious electrical stimulation of the hindpaw (left). FLI can be seen as punctate black dots concentrated in the superficial lamina. (B) Corresponding sections taken from a control animal in which electrodes were implanted into the hindpaw, but no footshock applied.

Inspection of PSTHs revealed that a substantial proportion (48/79, 61%) of DA neurones that were initially inhibited by noxious stimulation also exhibited a ‘rebound’ excitation following the period of inhibition (Figs. 3C, 5B and 6C). In 11/48 (23%), this manifested as a single excitation (Fig. 6C). However, the remaining neurones (37/48; 77%) showed repeated phases of excitation-inhibition, with gradually diminishing amplitude (Figs. 3C and 5B). Both types of rebound activity were characterized by precisely timed single spikes on successive trials, although a few single rebound cells (N⫽6) exhibited clusters of two to

three spikes. These clusters, however, did not conform to the standard criteria for bursts in DA neurones (Grace and Bunney, 1983). Significantly less rebound activity was observed in the initially excited DA neurones (one/13 (8%)) (␹2⫽10.58; df⫽1; P⬍0.01, Yate’s correction applied); this case consisted of a single inhibitory phase. Following work by Ungless et al. (2004), we observed that although the first component of the DA spike of inhibited DA neurones did have longer mean durations (1.5⫾0.03 ms) than excited neurones (1.4⫾0.1 ms), this difference was not statistically reliable (t⫽1.4, df⫽90,

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Fig. 2. Effects of three types of somatosensory stimulation on SC single unit (A–D) and multiunit activity (E, F): (A) Brushstroke applied to the hindpaw, at the times marked by a vertical dash, produced no evoked response. (B) Noxious pinch to the hindpaw (horizontal bar) produced a phasic increase in activity; scale as in A. (C) Raster plot and PSTH of 60 stimulations showing footshock-evoked activation (at the time indicated by the vertical dotted line) of a single SC neurone. (D) Location of nociceptive units in the SC reconstructed onto a section taken from the atlas of Paxinos and Watson (1997). (E) Location of multiunit recording sites in the SC. (F) Raster plot and PSTH of multiunit activation of the SC evoked by electrical stimulation (N⫽60) at the time indicated by the vertical dotted line. Abbreviations: DpG, deep gray layer; DpWh, deep white layer; InG, intermediate gray layer; InWh, intermediate white layer; Op, optic nerve layer; SuG, superficial gray layer; Zo, zonal layer.

P⬎0.05). Moreover, the initial durations of nigral DA spikes identified by standard criteria (Grace and Bunney, 1983) did not exhibit the bimodality reported for the VTA by Ungless et al. (2004). In our data, the initial phase of the DA spike was normally distributed with substantial overlap between the spikes of excited and inhibited neurones (Fig. 4). Although this parameter failed to differentiate the two response groups, inhibited cells had a significantly faster baseline firing rate (4.7⫾0.2 Hz) than excited cells (2.2⫾0.4; t⫽4.82, df⫽90 P⬍0.00001). Furthermore, the frequency of cells responding with excitation was higher in rostral SN (APⱕ5.3 mm; 9/33, 27.2%) than more caudal areas (APⱖ5.6 mm; 4/59, 6.8%) (␹2⫽7.325, df⫽1, P⬍0.0112) (Fig. 3B). No corresponding differences in re-

sponse type were found along the medio-lateral axis (␹2⫽2.5, df⫽1, P⬎0.05). Effects of intracollicular lidocaine on baseline and stimulus-evoked neural responses SC responses. Injections of lidocaine (N⫽21) into the SC suppressed both local baseline firing (measured by spike counts in the 50 ms prior to the application of footshock), and the phasic nociceptive response of the SC to footshock (measured by spike counts in the 50 ms post stimulation) (Fig. 5A and 5B). A two-way repeated measures ANOVA (factors DRUG [pre and post] and TIME [pre and post stimulation]) revealed significant main effects of TIME (F⫽28.8, df⫽1,

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Fig. 3. Nociceptive responses of putative DA neurones. (A) Examples of spike waveforms of DA neurones that were inhibited (left), excited (middle) or unresponsive (right) to noxious stimulation. (B) Location of recorded DA neurones (N⫽110), reconstructed onto sections taken from the atlas of Paxinos and Watson (1997). Black symbols represent inhibitory responses to the footshock (N⫽79, 72%), open symbols represent excitatory responses (N⫽13, 12%) and gray triangles represent non-responsive neurones (N⫽18, 16%). Bottom: An electrode tip position (marked by deposition of Pontamine Sky Blue; indicated by letter ‘E’) in the region of TH immunoreactivity. Abbreviations: SNPc substantia nigra pars compacta; SNPr, substantia nigra pars reticulata. (C) Responses of DA neurones inhibited (left) and excited (right) by noxious stimulation. Top: Both neurones types were unresponsive to non-noxious somatosensory brush-stroke. Middle: Both neurones were sensitive to noxious mechanical stimulation (pinch). Bottom: Responses to noxious footshock were also excitatory (left) or excitatory (right). The raster plots and PSTH were based on 60 trials with footshock applied at the time indicated by the vertical dotted line.

P⬍0.001) and DRUG (F⫽96.2, df⫽1, P⬍0.001). There was also a significant interaction between TIME and DRUG (lidocaine: F⫽11.1, df⫽1, P⬍0.05), indicating that the lidocaine

had a significantly greater influence on the phasic (poststimulation) nociceptive response than the baseline (prestimulation) firing (Fig. 5A and B).

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Fig. 4. Distribution of spike durations, measured from waveform onset to the peak of the first negativity (1st spike-component), for putative DA neurones that responded to noxious footshock with an initial inhibition (N⫽79) or initial excitation (N⫽13). Neurones to the right of dotted line exceed the additional electrophysiological criteria for identifying DA neurones suggested by Ungless et al. (2004).

DA responses. Of the 21 DA neurones recorded during SC suppression with lidocaine, 20 showed inhibitory responses to footshock prior to the injection: the remaining neurone was excited. Injections of lidocaine into the SC had comparatively minor damping effects on the nociceptive activity of DA neurones (Fig. 5C). This was confirmed in group analyses of the inhibitory responses which revealed a number of modest but statistically significant changes in DA nociceptive response characteristics (Table 1). There was a 28% decrease in the mean onset latency (t⫽5.1, df⫽19, P⬍0.00005) accompanied by a 13% increase in the mean duration (t⫽2.24, df⫽19, P⬍0.05) of the DA inhibitory response. The magnitude of the evoked suppression was reduced by about 20% following SC lidocaine (t⫽5.5, df⫽19, P⬍0.00005). These changes were accompanied by a 10% reduction in mean baseline firing rate (measured over the 500 ms prior to stimulation; t⫽2.4, df⫽19, P⬍0.05). This change in baseline firing was significantly correlated with changes in the latency (r⫽0.45; P⬍0.05) and inhibitory phase suppression (r⫽0.49; P⬍0.05) of evoked nociceptive responses. The single DA neurone displaying an excitatory response prior to SC lidocaine continued to show excitation following the injection, the amplitude of which was increased. Effects of intracollicular bicuculline on baseline and stimulus-evoked neural responses SC responses. Injections of bicuculline into the SC (N⫽19) produced local increases in both baseline and stimulus-evoked activity (Fig. 6A and B). Again a two-way repeated measures ANOVA (factors DRUG [pre and post] and TIME [pre and post stimulation]) revealed significant main effects of TIME (F⫽16.0, df⫽1, P⬍0.005) and DRUG

(F⫽6.6, df⫽1, P⬍0.05). The interaction between TIME and DRUG was also statistically reliable (F⫽11.1, df⫽1, P⬍0.05; bicuculline: F⫽6.6, df⫽1, P⬍0.05) again revealing a greater effect of the drug on the evoked response (Fig. 6A and B). Bicuculline-induced FLI extended over large areas of the SC (Fig. 6C), usually with some minor encroachment into the adjacent periaqueductal gray (PAG) and inferior colliculus. This suggests that during the period of the experiment bicuculline was exerting a widespread excitatory influence that affected much of the SC. DA responses. Of the 18 DA neurones recorded during SC activation with bicuculline, 15 showed an inhibitory response, three an excitatory response and one no response to footshock. Again, local activation of SC produced only modest effects on nociceptive responses of DA neurones (Fig. 6D). Unexpectedly, disinhibition of the SC with bicuculline also produced a reliable 23% decrease in the mean onset latency of inhibitory responses (t⫽4.18, df⫽14, P⬍0.0001; Table 2). However, the duration and magnitude of the evoked suppression were not significantly affected (t⫽0.5, df⫽14, P⬎0.05 and t⫽0.9, df⫽14, P⬎0.05 respectively). Although bicuculline elevated baseline firing of DA neurones (t⫽1.8, df⫽14, P⬍0.05), there was no significant correlation between this change and the change in response latency (r⫽0.18, P⬎0.05). The two neurones exhibiting excitatory nociceptive responses prior to injection continued to show excitation after the injection but with reduced onset latency and an increase in baseline firing. The DA neurone which was unresponsive prior to injection displayed an excitatory response after SC bicuculline. In summary, tonic suppression and activation of the SC produced respective decreases and increases in the base-

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Fig. 5. Effects of intracollicular lidocaine on local SC activity and nociceptive responses of DA neurones. (A) Baseline and stimulus-evoked multi-unit activity in the SC was suppressed by local injections of lidocaine. An individual example shows the decrease in spike-events during 50 ms pre-footshock (lower trace) and post-footshock (upper trace) counted over successive stimulus presentations. The dotted line shows when the injection of lidocaine was given. (B). Mean (⫾1 S.E.M.) multi-unit event counts in the SC, pre- and post-footshock, pre- and post-lidocaine (N⫽21). (C) Intracollicular lidocaine had modest effects on the nociceptive responses of DA neurones. Raster plots and PSTH of the responses of a DA neurone inhibited by footshock, before (left) and after (right) administration of lidocaine into the SC (dotted lines indicate when the footshock was delivered).

line firing of DA neurones. In addition, both manipulations of SC activity produced modest reductions in the onset latency of the evoked inhibitory response of DA neurones to footshock. The duration and magnitude of the DA inhibitory responses were only influenced by reductions of SC activity. Effect of collicular aspiration on nociceptive responses of DA neurones Since suppression of SC activity by lidocaine had only a small influence on the phasic nociceptive responses of DA neurones, we tested the effects of completely removing the ipsilateral SC Because the tectonigral projection is almost exclusively ipsilateral (Comoli et al., 2003), the SC was aspirated unilaterally. In all cases most of the SC was removed or damaged (along with the adjacent dorsolateral PAG and pretectum) (Fig. 7A). Twenty-three putative DA

neurones were successfully recorded following SC aspiration, all of which fulfilled the criteria of Grace and Bunney (1983), and all but two (21/23; 91%) that of Ungless et al. (2004). Of these 23 cells, 13 (57%) failed to respond to footshock, compared with 18/110 (16%) in intact animals (␹2⫽17.2, df⫽1, P⬍0.0001). The locations of DA neurones recorded following aspirations were similar to those in intact animals (Fig. 7B c.f. Fig. 3B). It is, therefore, unlikely that a difference in the location of recorded neurones could explain the different proportions of nociceptive neurones encountered. However, despite finding fewer nociceptive DA neurones, there was no difference in the proportion of excitatory and inhibitory responses encountered in lesioned and intact animals (␹2 [with Yate’s correction for small expected frequencies]⫽0.02, df⫽1, P⬎0.05). Similarly, no reliable differences in the inhibitory response characteristics of lesioned and intact animals were found (Table 3; Fig. 7C). Cells responding to footshock in le-

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Fig. 6. Effects of intracollicular bicuculline on local SC activity and nociceptive responses of DA neurones. (A) Baseline and stimulus-evoked multi-unit activity in the SC was facilitated by local injections of bicuculline. An individual example shows the increase in spike-events during 50 ms pre-footshock (lower trace) and post-footshock (upper trace) counted over successive stimulus presentations. The dotted line shows when the injection of bicuculline was given. (B). Mean (⫾1 S.E.M.) multi-unit event counts in the SC, pre- and post-footshock, pre- and post-bicuculline (N⫽18). (C) FLI in the SC following an injection of bicuculline (left). Plots of FLI reconstructed onto a central section of the SC taken from the atlas of Paxinos and Watson (1997) are shown for eight cases (right). (D) Intracollicular bicuculline also had modest effects on the nociceptive responses of DA neurones. Raster plots and PSTH of the responses of a DA neurone inhibited by footshock, before (left) and after (right) administration of bicuculline into the SC (dotted lines indicate when the footshock was delivered).

sioned animals also responded in the same way to pinch and remained unresponsive to brushstroke. In summary, despite almost complete destruction of the ipsilateral SC, a

normal pattern of nociceptive responses in DA neurones was observed, although fewer nociceptive cells were encountered.

V. Coizet et al. / Neuroscience 139 (2006) 1479 –1493 Table 1. Firing rate and characteristics of inhibitory responses of DA neurones to noxious footshock before and after intracollicular administration of lidocaine (N⫽20)

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the brain account for only a small proportion (⬃5%; Lacey et al., 1989) of the total. DA responses to noxious stimulation

Characteristic

Prelidocaine

Postlidocaine

Baseline firing (Hz) Onset latency (ms) Duration (ms) Mean suppression (Hz)

5.8⫾0.5 58.0⫾2.3 57.3⫾16.8 5.2⫾0.5

5.2⫾0.4* 41.5⫾2.6** 177.8⫾17.6* 4.2⫾0.5**

Mean⫾1 SEM. * P⬍0.05. ** P⬍0.0001.

DISCUSSION Technical considerations In the present study, the responses of both SC and DA neurones were recorded to three types of somatosensory stimulation: non-noxious brushstroke, noxious pinch and footshock. Unlike Yamasaki and Krauthamer (1990) who used a different anesthetic regimen, we observed no responses to non-noxious somatosensory stimuli either in the SC deep layers or DA neurones. There are several reasons for considering that the footshock used in the present study was frankly noxious: (i) The electrical stimulation parameters (2 ms, 3–5 mA) were based on previous work showing that stimulation of this intensity produces A␦ and C-fiber responses in the anesthetized rat spinal cord (Urch et al., 2003). (ii) The concentration of shock-evoked FLI in the medial part of the ipsilateral layers I and II of the lumbar spinal cord (Fig. 1) is the region specifically targeted by peripheral nociceptive afferents (Swett and Woolf, 1985; Besson, 1987). Interestingly, these layers also contain cells of origin of the spinotectal tract, (Verburgh et al., 1990). (iii) The observed pattern of evoked FLI in the spinal cord is consistent with that reported for noxious stimulation by others: footpinch and noxious heat (Hunt et al., 1987; Bullitt, 1990). (iv) The footshock evoked activity in nociceptive specific (high threshold) SC cells (Fig. 2) similar to that reported previously for noxious heat and mechanical stimuli (McHaffie et al., 1989; Redgrave et al., 1996a). (v) The footshock produced similar responses in putative DA neurones to those induced by a frankly noxious footpinch (Fig. 3). These considerations suggest we are reporting important aspects of pain processing both in the SC and ventral midbrain. A second technical issue concerns procedures for identifying putative DA neurones on the basis of electrophysiological criteria. Although the identity of present DA neurones cannot be confirmed with certainty, for the following reasons it is probably safe to assume that an overwhelming majority were DA. First, all cells met the identification criteria of Grace and Bunney (1983), and the overwhelming majority met the more stringent criterion suggested by Ungless et al. (2004). Secondly, an overwhelming majority was located in the TH immunoreactive region of SN pars compacta; corresponding to the A9 DA cell group (Lindvall and Bjorklund, 1974). Unlike the VTA (Ungless et al., 2004), non-DA neurones in this region of

DA neurones in the present study showed no evoked responses to non-noxious (brush-stroke) somatosensory stimulation. Phasic sensory responses to innocuous stimuli in DA neurones are not normally seen in anesthetized preparations, although tonic changes have been reported (Chiodo et al., 1980; Maeda and Mogenson, 1982). In contrast, a large majority of nigral DA neurones (84%) showed phasic responses to noxious mechanical stimulation (pinch). Of these, the majority (86%) responded with an initial inhibition and the remaining cells responded with an initial excitation. All pinch-responsive neurones also responded to noxious footshock with the sign of the response (inhibition or excitation), preserved. This mixture of inhibitory and excitatory responses, biased in favor of the former, fits well with the findings of previous studies in the rat (Tsai et al., 1980; Maeda and Mogenson, 1982; Mantz et al., 1989; Gao et al., 1990; Ungless et al., 2004). However, an advantage of using footshock is that the time of delivery of the noxious stimulation is precisely known, which enables neural response latencies to be calculated accurately. The short-latency nociceptive responses of DA neurones to footshock (inhibitions, 63.5 ms; excitations, 136.9 ms) were similar to those reported previously for SN DA neurones to trans-cutaneous noxious electrical stimulation applied to ‘different parts of the body’ (Gao et al., 1990). Importantly, the nociceptive latencies of DA neurones were substantially longer than those of SC neurones to the same stimulus (single unit: 9.9 ms; multiunit: 6.5 ms). This would be the case if the SC were responsible for providing DA neurones with afferent nociceptive information. The inhibitory responses of DA neurones to noxious footshock had significantly shorter latencies than their excitatory counterparts. A similar temporal relationship between inhibitory and excitatory responses has been reported by others (Tsai et al., 1980), but see Gao et al. (1990). However, the mean initial duration of DA spikes from waveform onset to the peak of the first negativity, (Ungless et al., 2004) of neurones exhibiting excitation was not reliably shorter than that of neurones exhibiting inhibitions. Indeed, this parameter was normally distributed in our data with no evidence of bimodality (Fig. 4). However, Table 2. Firing rate and characteristics of inhibitory responses of DA neurones to noxious footshock before and after intracollicular administration of bicuculline (N⫽15) Characteristic

Prebicuculline

Postbicuculline

Baseline firing (Hz) Onset latency (ms) Duration (ms) Mean suppression (Hz)

4.5⫾0.1 59.3⫾0.7 159.7⫾4.6 4.2⫾0.1

5.2⫾0.1* 45.3⫾0.5** 176.0⫾7.2 4.0⫾0.01

Mean⫾1 SEM. * P⬍0.05. ** P⬍0.001.

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Fig. 7. (A) A typical aspiration lesion of the SC reconstructed from photomicrographs of sections stained with Cresyl Violet. Dark gray shading indicates the regions completely removed, while light gray denotes damaged tissue. (B) Location of DA neurones (N⫽23) recorded following SC aspiration, reconstructed onto a section taken from Paxinos and Watson (1997). Black symbols represent inhibitory responses to footshock (N⫽9, 39%); the white symbol represents the excitatory response (N⫽1, 4%); the gray triangles represent non-responsive neurones (N⫽13, 57%); and the asterisk represents the location of the neurone whose data are illustrated in C. (C) Raster plot and PSTH of the responses of a DA neurone inhibited by footshock, after ipsilateral aspiration of the SC.

excited and inhibited cells were differentiated in terms of baseline firing rate and spatial location. The slower firing excited neurones were located more frequently in rostral pars compacta. Since the ascending nigrostriatal systems has a rostro-caudal topography (Maurin et al., 1999), it is possible that inhibited and excited compacta neurones could preferentially influence different striatal territories. However, although the neurochemical identity of the present excited cells remains uncertain, work in the VTA suggests that these cells may not be DA (Ungless et al., Table 3. Firing rate and characteristics of inhibitory responses of DA neurones to noxious footshock, in intact animals (N⫽79) and in animals with ipsilateral aspiration of the SC (and overlying cortex) (N⫽9) Characteristic

Intact

Collicular aspiration

Baseline firing (Hz) Onset latency (ms) Duration (ms) Mean suppression (Hz)

5.2⫾0.2 63.5⫾3.2 172.1⫾14.8 4.7⫾0.2

6.0⫾0.5 65.6⫾7.7 145.6⫾24.3 4.8⫾0.5

Mean⫾1 SEM.

2004). A second possibility is they may be part of a population of non-DA neurones that have been identified specifically in rostral SN in the guinea-pig and seem to be related more to the adjacent subthalamic nucleus (Overton et al., 1995). Immediately following the short-latency inhibitory nociceptive response, an excitatory offset rebound was observed in the majority of putative DA neurones. This rebound phenomenon has been reported by others following both footpinch and tailpinch (Tsai et al., 1980; Maeda and Mogenson, 1982), while Hommer and Bunney (1980) noted rebounds, some with gradually damping oscillatory periodicity, following sciatic nerve stimulation. However, on closer inspection our rebound ‘excitations’ usually consisted of single rather than a multi-spike excitations, as in a burst. DA neurones have been shown to be capable of producing bursts immediately after inhibitions provided an excitatory process is driving burst production (Overton et al., 1996). However, our observation of single spikes terminating the period of evoked-inhibition suggests that an excitatory opponent process (Daw et al., 2002; Ungless,

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2004) is not operating in the present circumstances. The opponent process hypothesis (Daw et al., 2002; Ungless, 2004) has been used to explain the paradoxical finding that, on the one hand, aversive stimuli increase DA levels in the forebrain of awake animals measured over a timescale of minutes by microdialysis (Abercrombie et al., 1989; Young, 2004), yet on the other, predominantly inhibitory nociceptive responses are observed on the millisecond timescale by electrophysiology. Without additional information, the present data cannot help to resolve this issue. The single spike rebounds in the present study produce apparent ‘excitations’ in PSTHs because they have a constant latency relative to stimulus onset. Since inhibitory responses to noxious stimuli in DA neurones appear to result from membrane hyperpolarization (Tsai et al., 1980), this could reset the slow oscillatory potential which underlies basal firing in these cells (Kang and Kitai, 1993). The fact that single spikes occur at a constant latency following the stimulus suggests that the period of inhibition which resets the pacemaker is extremely consistent (and probably has a rapid offset). However, in terms of influence on neurones in target structures, the effects of a presumed population response of inhibited DA neurones re-commencing to fire at approximately the same time, remains to be determined. Effects of collicular manipulations on the response of DA neurones to noxious stimulation Bicuculline injections, largely confined to the SC and probably causing excitation throughout much of the structure (Fig. 6C), increased both pre- and post-stimulus activity in the SC during noxious footshock. In contrast, intracollicular lidocaine injections decreased both baseline activity and the phasic response to noxious stimulation. These manipulations of SC activity had several effects on the responses of DA neurones inhibited by footshock. Both bicuculline and lidocaine decreased the latency of the DA nociceptive response, however, lidocaine also increased the duration but decreased the magnitude of the evoked suppression. In addition, both drugs caused respective changes in the baseline firing rate of DA neurones: bicuculline an increase, lidocaine a decrease. Although statistically significant, these effects were relatively small with evoked changes about 20% of pre-drug baselines. Although we had confirmation that much of the SC was affected by the bicuculline injections, no such assurance was available for lidocaine. The radius of the effective spread of 4% lidocaine has been estimated to be around 0.5 mm (Tehovnik and Sommer, 1997). This is similar to the separation distance of the injector and electrode in the present study. That the electrode may have been on the edge of the effective periphery of lidocaine suppression was indicated by our observations that baseline activity in the SC was normally above zero and the phasic response to the stimulus was never completely absent (Fig. 5A). Since the SC in the rat is approximately 2.5 mm rostrocaudal and medial–lateral (Paxinos and Watson, 1997), the lidocaine injections will almost certainly have affected only part of the structure. To overcome this limitation by

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producing a more complete suppression of SC activity, aspiration lesions were used to remove the entire structure ipsilateral to the DA recording electrode. Following this procedure, the number of nociceptive DA neurones encountered was significantly decreased; however, the nociceptive characteristics of DA neurones that remained sensitive to the noxious footshock (inhibited cells) were similar to those recorded in intact animals. A further puzzling issue in the present results is that effects of suppressing SC with lidocaine and aspiration lesions were different: the former led to changes in the response characteristics of DA inhibition and the latter did not. One possibility is that by suppressing activity in part of the SC, lidocaine may have led to an indirect excitation of surrounding tissue. The SC in most species, including monkey (Munoz and Istvan, 1998), cat (Rizzolatti et al., 1974) and ferret (Meredith and Ramoa, 1998), contains a tonically active lateral inhibitory network. Suppression of this tonic activity in one area of the SC by lidocaine may, therefore, have released adjacent areas from inhibition. Although speculative, the finding that both lidocaine and bicuculline produced some similar effects on DA nociceptive responses, suggests that their action within the SC may, at some level, have been similar. The observation of near normal nociceptive responses in animals with extensive ablations of the ipsilateral SC suggests that this structure is not a critical source of nociceptive input to DA neurones. However, it must be borne in mind that the SC lesions were unilateral, while the SC response to noxious stimuli is bilateral (Redgrave et al., 1996a; Telford et al., 1996). Although the direct projection from SC to the SN is almost exclusively ipsilateral (Comoli et al., 2003), hence the use of unilateral lesions in the present study, there are potential polysynaptic routes which cross midline by which nociceptive signals could be transmitted to DA neurones from the contralateral SC, including relays in the thalamus (Krauthamer et al., 1992; Krout et al., 2001) and pedunculopontine nucleus (Bolam et al., 1991; Steininger et al., 1992). However, evidence from a recent study in our laboratory suggests that primary nociceptive input to DA neurones probably arises from an alternative non-SC source, the parabrachial nucleus (Overton et al., 2005). This structure is the principal target of the spinomesencephalic pain pathway (Almeida et al., 2004; Klop et al., 2005). Thus, we have been able to demonstrate a direct anatomical projection from the parabrachial nucleus to SN pars compacta with complimentary anterograde and retrograde tract tracing techniques. Also, we have shown that nociceptive responses recorded from DA neurones in pars compacta are suppressed and in some cases abolished by injections of local anesthetic into the ipsilateral parabrachial nucleus. Functional implications Despite SC nociceptive responses having shorter latencies than those of DA neurones to the same stimulus, chemical modulations of SC activity had relatively minor effects on the responses of DA neurones to noxious footshock. Indeed, complete removal of the ipsilateral SC still

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left a significant population of DA neurones capable of exhibiting near normal responses to noxious stimulation. Collectively, these observations suggest that the SC can modulate the nociceptive responses of DA neurones, but does not supply the primary nociceptive sensory input. Thus, although the SC appears to be the critical relay for short-latency visual input to DA neurones (Dommett et al., 2005), the current results suggest it is unlikely to act as a general conduit for short-latency multisensory input. Rather, it appears that each modality may provide independent sensory information to DA neurones via separate anatomical circuits. Acknowledgments—Work was supported by the Wellcome Trust (068021 to P.O. and P.R.). E.D. was in receipt of a University of Sheffield studentship. The authors would like to thank Steven Clifford (Cambridge Electronic Design), Dr. Peter Furness for help with data analysis software, and Ms. Natalie Wood for histological assistance.

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(Accepted 26 January 2006) (Available online 3 March 2006)