Effects of baclofen and phaclofen on receptive field properties of rat whisker barrel neurons. Harold T. Kyriazi a, *, George E. Carvell b, Joshua C. Brumberg a, ...
BRAIN RESEARCH ELSEVIER
Brain Research 712 (1996) 325-328
Short communication
Effects of baclofen and phaclofen on receptive field properties of rat whisker barrel neurons Harold T. Kyriazi a, *, George E. Carvell b, Joshua C. Brumberg a, Daniel J. Simons " a Department of Neurobiology, School of Medicine, Biomedical Science Tower, University of Pittsburgh, Pittsburgh, PA 15261, USA b Department of Physical Therapy, School of Health and Rehabilitation Sciences, Unit,ersity of Pittsburgh, Pittsburgh, PA 15261, USA Accepted 12 December 1995
Abstract
Extracellular single-unit recordings were made in somatosensory cortical barrels of fentanyl-sedated rats. Whiskers were deflected singly or in paired combinations. Iontophoretically-applied (-)-baciofen disproportionately reduced weak responses, and phaclofen disproportionately increased them, resulting in more tightly focused or more broadly focused receptive fields, respectively. Both drugs had only minor effects on surround inhibition. In light of previous findings, we conclude that GABA A and GABAt~ mechanisms both act to enhance spatial contrast, but that the former plays a much greater role in enhancing temporal resolution. Keywords: y-Aminobutyric acid; Somatosensory; Cortex; Inhibition; Microiontophoresis
Inhibition generated within the somatosensory cortex plays an important role in shaping both temporal and spatial aspects of its responses to peripheral stimulation [1-5,10-14]. Previous studies have described the time course and spatial distribution of stimulus-evoked inhibition within the receptive fields of rat whisker/barrel neurons [7,19]. Inhibition appears to consist of a strong component lasting several tens of milliseconds and a weaker one lasting at least 1800 ms [15]. These latter findings suggest that barrel neurons are subject to both GABA Aand GABAB-mediated inhibition (e.g. see [9]). Recently, we demonstrated that, as in other species, bicuculline expands receptive fields and alters other aspects of barrel neuron response properties [16]. Here we quantify the effects of the GABA B agonist, baclofen, and antagonist, phaclofen (for review, see [6]). Adult female Sprague-Dawley rats were anesthetized with halothane, and a small opening was made in the cranium and dura overlying the right SmI vibrissa cortex. After completion of all surgical procedures, halothane was discontinued and the animals were narcotized with fentanyl, a synthetic opiate (5-10 p . g / k g / h , i.v.). Neuromuscular blockade was maintained with pancuronium bromide, and the rats were artificially respired via a tracheal cannula
* Corresponding author. Fax: (1) (412) 648-1441. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved
SSDI 0006-St~93(95)01 562-0
(for details, see [15]). The animal's condition was monitored by observation of femoral arterial blood pressure, tracheal air pressure, electroencephalogram, and pupillary reflexes. Extracellular single unit recordings in the layer IV barrels were made using 7-barrel glass microelectrodes containing an etched carbon fiber in the central barrel (see [16]). Microiontophoretic solutions consisted of 5 mM (-)-4-amino-3-(4-chlorophenyl)-butyric acid ( ( - ) b a c l o fen; Ciba-Geigy) in 0.9% NaC1, and 20 mM (RS)-3a m i n o - 2 - ( 4 - c h l o r o p h e n y l ) - p r o p y l phosphonic acid (phaclofen; Tocris Neuramin), also in 0.9% NaCI. Of the six microiontophoretic barrels, two contained baclofen, two phaclofen, one horseradish peroxidase (HRP; see below), and one saline for current balancing. Microiontophoretic currents ranged from 10-30 nA for baclofen and 10-65 nA for phaclofen (respective means and standard deviations of 20.0 + 8.4, n = 6, and 29.2 + 14.0, n = 19), and were adjusted to produce a noticeable change in spontaneous activity, assessed qualitatively by audio monitoring or quantitatively by computer program. 65 nA was the largest current that could be ejected, using two barrels simultaneously. Retaining currents were - 1 5 nA. Individual whiskers were deflected by electromechanical stimulators [18], and single units were distinguished by means of an amplitude discriminator. Selected recording locations were marked by iontophoretic release of HRP [20]. Experiments were terminated by injection of barbitu-
H.T. Kyriazi et al. / Brain Research 712 (1996) 325-328
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rate, and the brains were perfused for histological processing [17]. Units were included if they were found, upon histological reconstruction, to be located within the cytochrome oxidase-rich, layer IV barrels. All recordings were obtained from regular-spike units (RSUs), which are presumed, in the barrels, to be spiny stellate cells (see [19]). Data were obtained from 3 animals, which were part of a larger study examining the effects of the GABA A antagonist bicuculline methiodide (BMI) [16]. Fig. 1 shows the effects of baclofen and phaclofen on the responses of a single barrel neuron to deflections of its principal whisker (PW) and of the caudally adjacent one. The PW is defined as the vibrissa that most strongly excites the neuron, and which is subsequently found to correspond anatomically to the barrel in which the cell resides. Pre-stimulus activity was altered the most, being completely eliminated by baclofen (30 nA) and increased 59% by phaclofen (32 nA). ON and OFF responses evoked by PW stimulation were largely unchanged in the presence of the antagonist, whereas with phaclofen, they increased 30% and 31%, respectively. The less vigorous responses evoked by adjacent whisker deflections were more strongly affected by the drugs (see insets in Fig. 1). As with spontaneous activity, baclofen completely eliminated the response, and phaclofen caused a 57% increase. All of the neurons studied with baclofen (n = 6) tended to experience across-the-board decreases, but in 3 instances a small increase (range of 6-24%) in a PW ON or OFF response was observed. Phaclofen's effects were more consistent.
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Time (ms) Fig. 1. Effects of baclofen and phaclofen on principal and adjacent whisker responses. PSTHs show the cumulative responses of a single unit when its PW was rapidly deflected at one of 8 angles (first solid arrow), held for 200 ms, and then returned to its resting position (second solid arrow). Whisker stimulators were placed 10-12 mm from the skin surface, and deflection amplitude was 1.0 mm. This unit was recorded at a depth 812 /xm below the pial surface, and was found to be located in the D2 barrel. Each histogram shows the summed response to 80 stimuli (10 repetitions at each of the 8 directions). The caudally adjacent whisker (D1) was also deflected (open arrows show onset and offset), but its response, small to begin with, was greatly diminished by the inhibition evoked by immediately prior PW deflection. Insets show the caudal whisker ON responses (when not preceded by PW deflection) at the same scales as the other PSTHs.
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Fig. 2. Effects of baclofen and phaclofen on average response magnitude. A: plotted are the means + S.E.M. of responses to all eight deflection angles (expressed in terms of spikes occurring in a 20 ms response window), and spontaneous activity. Paired t-test P values: < 0.1, * ; < 0.05, * *, < 0.01, • * * ; not significant ( > 0.1), N.S. B: ratios of drug to pre-drug average response magnitudes plotted against average initial magnitudes. N values are the same as in Panel A. The nine points represent maximal and average ON and OFF responses of principal and caudally adjacent whiskers, and spontaneous activity, which is the leftmost point in each graph. PW ON and OFF responses at the best deflection angle are the two rightmost points. There are no error bars because the y-values are the ratios of means, rather than the means of ratios.
H.T. Kyriazi et al. / Brain Research 712 (1996) 325-328
Of the 26 PW ON or OFF responses obtained from the 13 phaclofen units, all but two increased (4-261%); the two decreases were 5% and 13%. The smaller, adjacent whisker responses also were consistently affected by phaclofen, as only 4 of 26 ON or OFF responses failed to increase. Fig. 2 shows quantitative data for all of the studied units. Phaclofen produced significant increases in spontaneous activities and in responses evoked by deflecting the PW or its caudally adjacent neighbor. Baclofen produced a robust decrease in spontaneous activity and in responses evoked by the adjacent whisker; effects on PW-evoked responses were smaller and more variable. Because of the relatively small numbers of units studied, some control values differ between the baclofen and phaclofen data sets (especially the PW OFF responses and spontaneous activities). Nevertheless, the changes effected by each drug (increases or decreases in responsiveness) were consistent. Moreover, for both drug conditions, a consistent trend was observed, such that initially smaller activities were more greatly affected. This is shown in Fig. 2B, which plots the relative changes in the average responses evoked by each drug as a function of the magnitude of the initial activity. Barrel neurons respond most vigorously to deflections of their PW and to a lesser and more variable extent to adjacent whiskers. We computed a measure of the focus of each neuron's receptive field by taking the ratio of its caudally adjacent whisker ON response to that of its PW; lower values indicate tighter focus. (The inverse ratio would be preferred because it increases as focus increases, but it occasionally gives infinite values.) The mean value, of 0.22 (Fig. 3A), is close to the value of 0.19 obtained previously under similar conditions (see [19]). Baclofen reduced this ratio to 0.074, because it decreased the (smaller) adjacent whisker responses more than the (larger) PW responses. This finding echoes those of previous studies in cat primary somatosensory cortex [13,14]. Phaclofen had opposite effects, producing somewhat more broadly focused receptive fields (0.245 + 0.030 vs. 0.216 + 0.026;
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n = 19). By comparison, BMI added to a subset of the same neurons produced a much greater field broadening (0.480 ___0.152 vs. 0.213 + .030, n = 10). Given the large effect with baclofen, we attribute this difference more to the low binding affinity of phaclofen (see [6]) than to the 3-to-1 preponderance of GABA A to GABA B receptors in layer IV of rat parietal cortex [8]. Effects of baclofen and phaclofen on an inhibitory receptive field characteristic also were examined. When deflection of the PW follows an adjacent whisker movement by 20-30 ms, the PW ON response generally is reduced by about half [19]. The ratio of the magnitude of this reduced, 'conditioned test' response to that of its 'test alone' value is called 'the condition-test ratio'; we take this as an inverse measure of the strength of surround inhibition (see Fig. 3B legend). Fig. 3B shows that baclofen and phaclofen exert only small, if any, effects on surround inhibition. This study reports the effects of (-)-baclofen and phaclofen on regular-spike barrel units, whose behavior in the presence of BMI, as well as GABA, was reported previously [16]. The effects of BMI and phaclofen are qualitatively similar, in that both produce proportionally greater enhancement of initially smaller responses. Similarly, baclofen and GABA disproportionately reduce weaker responses, thus enhancing spatial resolution. The study with BMI indicated that GABA A receptor mechanisms play a major role in mediating the time-dependent inhibition observed when whiskers are deflected in rapid succession. By contrast, the GABA~ effectors employed in the present study had little or no effect on surround inhibition despite their efficacy in otherwise influencing overall neuronal excitability. Taken together, these findings support the conclusions of Connors et al. [9] that GABA A receptors are the primary mediators of fine temporal resolution, while GABA B receptors, operating with a much slower timecourse, act with GABAA-mediated inhibition to decrease neuronal excitability, and thus enhance spatial contrast.
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Fig. 3. Effect of baclofen and phaclofen on excitatory (A) and inhibitory (B) receptive field properties. For baclofen, n = 6; for phaclofen, n = 19. Paired t-test P values: < 0.1, * ; < 0.01, " * * ; not significant ( > 0.1), N.S. A: 'receptive field focus' is the ratio of the unit's caudally adjacent whisker ON response at all 8 angles to that of its PW. B: the strength of surround inhibition is measured by the condition-test ratio, for which a value of 1.0 indicates no response suppression, and a value of 0.0 corresponds to complete suppression.
[1] Alloway, K. and Burton, H., Bicuculline-induced alterations in neuronal responses to controlled tactile stimuli in the second somatosensory cortex of the cat: a microiontophoretic study, Somatosens. Res., 3 (1986) 197-211. [2] Alloway, K. and Burton, H., Differential effects of GABA and bicuculline on rapidly- and slowly-adapting neurons in primary somatosensory cortex of primates, Exp. Brain Res., 85 (1991) 598-610. [3] Alloway, K., Rosenthal, P. and Burton, H., Quantitative measurements of receptive field changes during antagonism of GABAergic transmission in primary somatosensory cortex of cats, Exp. Brain Res., 78 (1989) 514-532. [4] Batuev, A., Alexandrov, A. and Scheynikov, N., Picrotoxin action on the receptive fields of the cat sensorimotor cortex neurons, .I. Neurosci. Res., 7 (1982) 49-55.
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[5] Batuev, A., Alexandrov, A., Scheynikov, N., Kcharazia, V. and An, C., The role of inhibitory processes in the formation of functional properties of neurons in vibrissal projection zone of the cat somatosensory cortex, Exp. Brain Res., 76 (1989) 198-206. [6] Bowery, N., GABAa receptors and their significance in mammalian pharmacology, Trends Pharmacol. Sci., 10 (1989) 401-407. [7] Carvell, G. and Simons, D., Membrane potential changes in rat Sml cortical neurons evoked by controlled stimulation of mystacial vibrissae, Brain Res., 448 (1988) 186-191. [8] Chu, D., Albin, R., Young, A. and Penney, J., Distribution and kinetics of GABA a binding sites in rat central nervous system: a quantitative autoradiographic study, Neuroscience, 34 (1990) 341357. [9] Connors, B., Malenka, R. and Silva, L., Two inhibitory postsynaptic potentials, and GABA A and GABA B receptor-mediated responses in neocortex of rat and cat, J. Physiol., 406 (1988) 443-468. [10] Dykes, R., Landry, P., Metherate, R. and Hicks, T., Functional role of GABA in cat primary somatosensory cortex: shaping receptive fields of cortical neurons, J. Neurophysiol., 52 (1984) 1066-1093. [11] Gardner, E. and Costanzo, R., Temporal integration of multiple-point stimuli in primary somatosensory cortical receptive fields of alert monkeys, J. Neurophys., 43 (1980) 444-468. [12] Hicks, T. and Dykes, R., Receptive field size for certain neurons in primary somatosensory cortex is determined by GABA-mediated intracortical inhibition, Brain Res., 274 (1983) 160-164.
[13] Kaneko, T. and Hicks, T., Baclofen and 3,-aminobutyric acid differentially suppress the cutaneous responsiveness of primary somatosensory cortical neurones, Brain Res. 443 (1988) 360-366. [14] Kaneko, T. and Hicks, T., GABAs-related activity involved in synaptic processing of somatosensory information in S1 cortex of the anaesthetized cat, Br. J. Pharmacol. 100 (1990) 689-698. [15] Kyriazi, H., Carvell, G. and Simons, D., OFF response transformations in the whisker/barrel system, J. Neurophys., 72 (1994) 392401. [16] Kyriazi, H., Carvell, G., Brumberg, J. and Simons, D., Quantitative effects of GABA and bicuculline methiodide on receptive field properties of neurons in real and simulated whisker barrels, J. Neurophys., in press [17] Land, P. and Simons, D., Cytochrome oxidase staining in the rat Sml barrel cortex, J. Comp. Neurol., 238 (1985) 225-235. [18] Simons, D., Multi-whisker stimulation and its effects on vibrissa units in rat Sml barrel cortex, Brain Res., 276 (1983) 178-182. [19] Simons, D. and Carvell, G., Thalamocortical response transformation in the rat vibrissa/barrel system, J. Neurophysiol., 61 (1989) 311-330. [20] Simons, D. and Land, P., A reliable technique for marking the location of extracellular recording sites using glass micropipettes, Neurosci. Lett., 81 (1987) 100-104.
