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Classification of inhibitory responses of the hamster gustatory cortex
BRAIN RESEARCH ELSEVIER
Brain Research 666 (1994) 270-274
Short communication
Classification of inhibitory responses of the hamster gustatory cortex Jill A. L o n d o n a,*, Richard G. Wehby b a Department of BioStructure and Function, MC3705, University of Connecticut Health Center, Farmington, CT06030, USA b Center for Neurological Sciences, University of Connecticut Health Center, Farmington, CT06030, USA Accepted 20 September 1994
Abstract
Neuronal activity was recorded in the gustatory cortex of the golden Syrian hamster in response to application of taste stimuli to the anterior tongue. Two classes of inhibitory responses were detected: (i) a decrease in activity in response to application of individual taste stimuli: and (2) a decrease in activity in response to application of a mixture of taste stimuli but not in response to application of individual taste stimuli.
Keywords: Gustatory cortex; Hamster; Inhibition; Extracellular activity; Taste cortex; Taste stimuli Many reports of neuronal activity in the gustatory cortex of the rodent describe and classify neurons which are excited by application of chemosensory stimuli [4,5,6,8,11,12,13]. Inhibitory responses have also been observed in the gustatory cortex of several mammalian species, e.g. the monkey [9], the dog [2], the hamster [11], and the rat [6,12]. In the rat gustatory cortex, the percentage of inhibitory responses to total responses in cortical gustatory areas has been reported at 27% [12], 30-42% [8] and as high as 70% [6]. Despite the frequency of occurrence of these inhibitory responses, a description of the types of inhibition has not yet been reported. A complete understanding of the processing of taste information in the cortex cannot be realized without considering both the excitatory and inhibitory responses of neurons in the cortex. In this report, we describe two types of inhibitory responses in the hamster gustatory cortex that occur in response to application of taste stimuli: (1) inhibition resulting from application of a single taste stimulus; (2) inhibition resulting from application of a mixture of taste stimuli but not the components of the mixture. Adult, male, golden Syrian hamsters (Mesocricetus auratus) were initially anesthetized with Nembutal (80 m g / k g , i.p.) and maintained with urethane (425 m g / k g , i.p.). Atropine (1 m l / k g , i.p.) was administered one hour prior to injection of urethane. The animal was
* Corresponding author. Fax: (1) (203) 679-2910. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 1 1 3 - 3
placed in a non-traumatic head-holder [1] and rotated 90 ° so that the insular cortex was pointed up. A craniotomy was performed and the dura resected (approximately 1-5 mm anterior to bregma, and 5 mm lateral to the midline) leaving approximately a 7 × 5 m m opening over the cortex. Single- or multi-unit activity was recorded with glass micropipettes (1.0-1.5 Mg/, 0.5 M NaC1) inserted perpendicular to the surface of the cortex. The signals were fed to an extracellular amplifier (Dagan) and observed with an oscilloscope (Tektronix). Data was also fed into a pulse conditioner (Biomedical Systems) and from there into a V C R (Panasonic) for storage on tape. An oscilloscope camera (Tektronix) was used to make hard copies of the data. Data was also scanned (Hewlett Packard) into a computer (Gateway) for subsequent analysis and storage. To characterize the activity, multi- or singleunit recordings were made of neuronal activity before, during and after presentation of stimuli. Electrode penetrations were made in a grid-like fashion, the interval between penetrations was typically 50 /xm apart. In some experiments, the interval between penetrations was 200-300 ~ m apart. The single component chemical stimuli were: 0.1 M NaCl, 0.3 M KCI or 0.3 M sucrose, and the mixture solution contained the three component stimuli at the same final concentrations [7]. Stimuli were washed off with distilled water. Sapid stimuli and washes were delivered at room temperature (22°C). Stimulus solutions were delivered at approximately 0.5 m l / s from a gravity flow system and
J.A. London, R.G. Wehby/ Brain Research 666 (1994) 270-274
were confined to the anterior tongue by a plastic dam. Tactile stimuli were applied by lightly stroking or tapping a paint brush across the taste field. Thermal stimuli (either 40°C or 10°C distilled water) were applied in the same manner as the sapid stimuli. The following data taking paradigm was employed: 2-60 s of baseline activity, followed by 1-60 s of stimulus application, followed by 120 s of recovery. In some experiments, distilled water was applied for 50-60 s prior to stimulus application. A neuron was considered to have an inhibitory response if, upon application of the stimulus, the activity decreased by 2 standard deviations from baseline activity. In order to recover the location of the activity in the cortex, horseradish peroxidase (HRP) (Sigma) was iontophoresed at locations where taste activity was recorded (3-7 s, 200 ms pulse, 50% duty cycle, 2/xA). At the end of the experiment, the animals were anesthetized and perfused. Their brains were removed and placed in a 30% sucrose buffer solution. Frozen sections were cut at 40 /xm on a sliding microtome. The H R P was visualized by the Hanker-Yates method [3] and counterstained with thionin. Reaction product spots were typically < 100/xm in diameter. Slides were examined with a light microscope (Leitz), and the reaction products located by visual inspection. Photographs and camera lucida drawings of the spot locations were made. Neurons which were either excited or inhibited in response to application of taste stimuli to the anterior tongue were located throughout the dysgranular/ agranular insular cortex (n = 91 animals). Of the 1,489 taste-responsive neurons identified, 1,147 neurons were excited and 342 neurons were inhibited by application of chemical stimuli. Data describing the excitatory neuronal activity will be presented in another paper (London and Wehby, in preparation). There were at least two classes of neurons inhibited by application of chemical stimuli to the anterior tongue: (1) single-component-inhibited neurons: neurons inhibited by only one of the component stimuli; and (2) mixture-inhibited neurons: neurons inhibited by the mixture, but not any single component stimulus alone. Single-component-inhibited neurons. There were a substantial number of neurons (n = 295) that were inhibited by application of a single stimulus. The activity of these neurons was not affected by application of the other chemical stimuli, nor by thermal or tactile stimulation. For 59 of these neurons the degree of inhibition observed was greater for single component stimuli than for the mixture stimulus, suggesting an interaction of the component stimuli (data not shown). These neurons fall into three sub-categories. The first such sub-category contains neurons inhibited solely by application of NaC1 (n = 102 neurons) (Fig. 1). In this experiment, the activity of one spontaneously active
27t
NaCl
Sucrose
KCI I_I.I. LI
L.I___I I _ I _ I
[l I ]I l-l-l-l
I__
L._ILI~_U lilt_IlL_
l-l-l---I
II III U-II-III "
50 ~,v I see
Fig. 1. Response of one cortical neuron to application of three component stimuli. This neuron was inhibited by application of NaC1 to the anterior tongue. Up arrowhead marks start of application of stimulus, down arrowhead marks start of water rinse. Stimuli were applied for 3 s.
neuron was recorded and was inhibited by application of NaCl. Weaker concentrations of NaC1 would diminish, but not block spontaneous activity (data not shown). This neuron had a spontaneous firing rate of 6.0 spikes/s. A second sub-category contains neurons inhibited by application of sucrose (n = 92 neurons) (Fig. 2). In the recording shown, two neurons were identified which were inhibited by application of sucrose. The spontaneous firing rate of the neuron with the larger spike was 1.1 spikes/s. The spontaneous firing rate of the neuron with the smaller spike was 4.2 spikes/s. The third sub-category identified contains
NaCl
Sucrose
KC1
20 # V
0.5 s e c Fig. 2. Responses of two simultaneously recorded cortical neurons to application of three component stimuli. Both neurons were inhibited by application of sucrose to the anterior tongue. Stimuli were applied for 2.5 s. Symbols are the same as for Fig. 1.
272
J.A. London, R.G. Wehby/Brain Research 666 (1994) 270-274
neurons inhibited solely by application of KC1 (n --- 101 neurons) (Fig. 3). The neuron shown here had a spontaneous firing rate of 14.7 spikes/s. Mixture-inhibited neurons. There were 47 neurons that were inhibited only when the mixture solution was applied, but not when the individual stimuli were applied (Fig. 4). The spontaneous firing rate of the neuron in this figure is 5.9 spikes/s. Inhibitory responses comprise a significant proportion of responses recorded in the gustatory cortex of the hamster. Approximately 23% of the neurons that responded to application of taste stimuli were inhibited by this application (342/1147). This value is in agreement with that reported by several other investigators for neurons in the rat cortex. For example, Yamamoto et al. [12] reported that 27% (29/111) of the neurons affected by application of taste stimuli in the rat dysgranular cortex were inhibited. Ogawa et al. [8], reported that 41.2% (21/51) of the neurons in the granular insular cortex, and 29.2% (7/24) of the neurons in the dysgranular insular cortex were inhibited by either one or two stimuli. One group of investigators has reported a frequency of inhibitory responses for a subset of cortical taste neurons at 70% [7]. However, only 30% of these neurons actually exhibited a significant decrease in firing rate [7]. We conclude that taste responsive neurons in the hamster gustatory cortex have a similar distribution of taste responsive neurons as that of the rat gustatory cortex. Recordings of inhibitory responses were made throughout the course of an experiment, and were intermixed with recordings of excitatory responses. Furthermore, in other experiments utilizing the same preparation and stimulus paradigm, EKG and evoked
NaCI
Sucrose
KCI
50~v I 0.5 s e c Fig. 3. Response of a cortical neuron that is inhibited by application of KCI to the anterior tongue. In this case, activity was almost, but not completely inhibited. Application of other stimuli did not effect the activity of the cell. Stimuli were applied for 1.5 s. Symbols are the same as for Fig. 1.
NaCI
Stlcros ~.
KCI
Mixtur,
50 uV 2 sec Fig. 4. Response of a cortical neuron inhibited by application of mixture solution only. There was negligible effect of application of the three component stimuli. Stimuli were applied for 3 s. Symbols are the same as for Fig. 1.
potential recordings before, during and after stimulus application were indistinguishable (unpublished observations). These observations argue against the possibility that the inhibitory responses result from an arousal-state change of the animal, but reflect neuronal activity that occurs in response to application of taste stimuli. The sharpness of tuning of the inhibitory responses is much greater than seen in the rat cortex. The rat granular and dysgranular gustatory cortices contain neurons inhibited by a single stimulus, as well as neurons inhibited by more than one stimulus [8,12]. This difference may be accounted for by three factors. One factor may be that there is a species difference between the rat and the hamster. The distribution of taste bud fields in the oral cavity differs for rat and hamsters. In addition, the hamster has evolved in a very different ecological niche than the rat. As a consequence, the neuronal response to the same stimuli may be different. A second possibility is that there is a difference in the response properties of neurons from different parts of the gustatory cortex. Our recordings were made in the agranular/dysgranular region, whereas the other investigators recorded responses in the granular/dysgranular region. A final reason why
J.A. London, R.G. Wehby / Brain Research 666 (1994) 270-274 Table 1 Distribution of inhibitory responses Neuron type
Number of neurons
Range of spontaneous activity (spikes/s)
Average firing rate (spikes/s)
NaCI KCI Sucrose Mixture
102 101 92 47
0.42-10.1 0.42-16.7 0.35- 9.8 0.44-12.5
7.0 + 0.23 7.3 + 0.36 5.2-t-0.14 6.8 _+0.43
the neurons in the agranular region may be more tightly tuned, and potentially the most interesting reason, is that stimulation in this report was confined to the anterior tongue while other investigators have used whole mouth stimulation. It is possible that excitatory a n d / o r inhibitory inputs from other peripheral taste fields, such as the soft palate or glossopharyngeal fields, converge on taste responsive neurons in the agranul a r / d y s g r a n u l a r cortex, in order to blend taste information from the different peripheral taste fields. This 'mixed input' may serve to sharpen the ability of the animal to detect different stimulus types. Differences between the spontaneous activity of sucrose-inhibited neurons and the other neuron types was recorded (Table 1). The range of spontaneous activity recorded for all categories of the inhibitory response neurons was 0.35-16.7 spikes/s. The average spontaneous firing rates for all the inhibitory response neurons was 5.2 + 0.14 to 7.0 + 0.23 spikes/s. Comparisons of the mean spontaneous firing rates for neurons inhibited by application of NaC1, KC1 or the mixture stimulus (ranging from 6.8 + 0.43 to 7.0 + 0.23 spikes/s) were not significantly different (NaC1 compared to KC1, z = 0.21; NaC1 compared to mixture, z = 0.13, KCI compared to mixture, z = 0.26). However, the mean spontaneous firing rate of sucrose-inhibited neurons (5.2 + 0.14 s p i k e s / s ) was significantly different when compared to the m e a n spontaneous firing rate for all the other neurons in the three cell types (z = 3.29, P < 0.001) with the NaCl-inhibited neurons exhibiting the biggest difference (z = 3.2, P < 0.001). This difference in spontaneous activity of the sucrose-inhibited neurons from the other neurons suggests the possibility of a division, based on neuronal response properties, of cortical activity into hedonic and nonhedonic streams of information. All neurons inhibited by sapid stimuli were located in the d y s g r a n u l a r / a g r a n u l a r insular cortex. While there did not seem to be a strict chemotopic organization in which neurons of one type were restricted to a particular region of the cortex, there did seem to be clusters of like-responsive neurons; a cluster consisted of two to three like-responsive neurons located within 50 /~m of each other (Fig. 2 for example). In 18 animals, clusters of neurons inhibited by application of
273
NaCI were recorded. Often the activities of these neurons were recorded simultaneously. In animals in which two to four clusters were recorded in the same animal (n = 10 animals), clusters were located 100-200 /xm apart. Similar to the neurons that were inhibited by NaCI application, two to three clusters containing two to three sucrose-inhibited neurons were recorded in the same animal (n = 9 animals). These clusters were also located 100-200 /~m apart. Comparable clusters containing two to three KCl-inhibited neurons were also recorded at 1 0 0 - 2 0 0 / x m intervals (n = 9 animals). While component specific neurons appeared in groups, mixture-inhibited neurons were located throughout the agranular insular cortex and did not appear to be clustered within a penetration, or adjacent penetrations. They also did not appear adjacent to a particular class of single-component-inhibited neuron. Our results indicate that the processing of gustatory information is more complex than had recently been thought. There are several types of inhibitory neurons in the gustatory cortex of the hamster. Single-component inhibited-neurons appear clustered together in this cortical region. The role these inhibitory responses may play in the processing of taste information remains to be determined. Some of this work has appeared in abstract form [10]. This work was supported by P50DC00168 and 5T32DC00025. The authors would like to thank Drs. Marion Frank and Joel Zeiger for their critical comments on the manuscript and Mr. Yi-Long Sun for some of the earlier work. [1] Erickson, R.P., Nontraumatic headholders for mammals, Physiol. Behac., 1 (1966) 97-98. [2] Funakoshi, M. and Ninomiya, Y., Relations between the spontaneous firing rate and taste responsiveness of the dog cortical neurons, Brain Res., 262 (1983) 155-159. [3] Hanker, J.S., Yates, P.E., Metz, C.B. and Rustioni A., A new specific, sensitive and non-carcinogenic reagent for the demonstration of horseradish peroxidase, Histochem. J., 9 (1977) 789792. [4] Kosar, E., Grill, H.J. and Norgren, R., Gustatory cortex in the rat. I. Physiological properties and cytoarchitecture, Brain Res., 379 (1986) 329-341. [5] Kosar, E., Grill, H.J. and Norgren, R., Gustatory cortex in the rat. II. Thalamocortical projections, Brain Res., 379 (1986) 342352. [6] Kosar, E. and Schwartz, G.J., Cortical unit responses to chemical stimulation of the oral cavity in the rat, Brain Res., 513 (1990) 212-224. [7] McPheeters, M., Hettinger, T.P.~ Nuding, S.C., Savoy, L.D., Whitehead, M.C. and Frank, M.E., Taste-responsive neurons and their locations in the solitary nucleus of the hamster, Neuroscience, 34 (19990) 745-758. [8] Ogawa, H., Murayama, N. and Hasegawa, K., Difference in receptive field features of taste neurons in rat agranular and dysgranular insular cortices, Exp. Brain Res., 91 (1992) 408-414. [9] Scott, T.R., Plata-Salaman, C.R., Smith, V.L. and Giza, B.K., Gustatory neural coding in the monkey cortex: stimulus intensity, J. Physiol., 65 (1991) 76-86.
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[10] Wehby, R.G. and London, J.A., Inhibition of neuronal responses in the hamster gustatory cortex, Chem. Senses, 17 (1992) 716. [11] Yamamoto, T. and Kitamura, R., A search for the cortical gustatory area in the hamster, Brain Res., 510 (1990) 309-320. [12] Yamamoto, T., Yuyama, N., Kato, T. and Kawamura, Y., Gusta-
tory responses of cortical neurons in rats. I. Response characteristics, J. Neurophysiol., 51 (1984) 616-635. [13] Yamamoto, T., Yuyama, N., Kato, T. and Kawamura, Y., Gustatory responses of cortical neurons in rat. II. Information processing of taste quality, Z Neurophysiol., 53 (1985) 1356-1369.
Brain Research 666 (1994) 270-274
Short communication
Classification of inhibitory responses of the hamster gustatory cortex Jill A. L o n d o n a,*, Richard G. Wehby b a Department of BioStructure and Function, MC3705, University of Connecticut Health Center, Farmington, CT06030, USA b Center for Neurological Sciences, University of Connecticut Health Center, Farmington, CT06030, USA Accepted 20 September 1994
Abstract
Neuronal activity was recorded in the gustatory cortex of the golden Syrian hamster in response to application of taste stimuli to the anterior tongue. Two classes of inhibitory responses were detected: (i) a decrease in activity in response to application of individual taste stimuli: and (2) a decrease in activity in response to application of a mixture of taste stimuli but not in response to application of individual taste stimuli.
Keywords: Gustatory cortex; Hamster; Inhibition; Extracellular activity; Taste cortex; Taste stimuli Many reports of neuronal activity in the gustatory cortex of the rodent describe and classify neurons which are excited by application of chemosensory stimuli [4,5,6,8,11,12,13]. Inhibitory responses have also been observed in the gustatory cortex of several mammalian species, e.g. the monkey [9], the dog [2], the hamster [11], and the rat [6,12]. In the rat gustatory cortex, the percentage of inhibitory responses to total responses in cortical gustatory areas has been reported at 27% [12], 30-42% [8] and as high as 70% [6]. Despite the frequency of occurrence of these inhibitory responses, a description of the types of inhibition has not yet been reported. A complete understanding of the processing of taste information in the cortex cannot be realized without considering both the excitatory and inhibitory responses of neurons in the cortex. In this report, we describe two types of inhibitory responses in the hamster gustatory cortex that occur in response to application of taste stimuli: (1) inhibition resulting from application of a single taste stimulus; (2) inhibition resulting from application of a mixture of taste stimuli but not the components of the mixture. Adult, male, golden Syrian hamsters (Mesocricetus auratus) were initially anesthetized with Nembutal (80 m g / k g , i.p.) and maintained with urethane (425 m g / k g , i.p.). Atropine (1 m l / k g , i.p.) was administered one hour prior to injection of urethane. The animal was
* Corresponding author. Fax: (1) (203) 679-2910. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 1 1 3 - 3
placed in a non-traumatic head-holder [1] and rotated 90 ° so that the insular cortex was pointed up. A craniotomy was performed and the dura resected (approximately 1-5 mm anterior to bregma, and 5 mm lateral to the midline) leaving approximately a 7 × 5 m m opening over the cortex. Single- or multi-unit activity was recorded with glass micropipettes (1.0-1.5 Mg/, 0.5 M NaC1) inserted perpendicular to the surface of the cortex. The signals were fed to an extracellular amplifier (Dagan) and observed with an oscilloscope (Tektronix). Data was also fed into a pulse conditioner (Biomedical Systems) and from there into a V C R (Panasonic) for storage on tape. An oscilloscope camera (Tektronix) was used to make hard copies of the data. Data was also scanned (Hewlett Packard) into a computer (Gateway) for subsequent analysis and storage. To characterize the activity, multi- or singleunit recordings were made of neuronal activity before, during and after presentation of stimuli. Electrode penetrations were made in a grid-like fashion, the interval between penetrations was typically 50 /xm apart. In some experiments, the interval between penetrations was 200-300 ~ m apart. The single component chemical stimuli were: 0.1 M NaCl, 0.3 M KCI or 0.3 M sucrose, and the mixture solution contained the three component stimuli at the same final concentrations [7]. Stimuli were washed off with distilled water. Sapid stimuli and washes were delivered at room temperature (22°C). Stimulus solutions were delivered at approximately 0.5 m l / s from a gravity flow system and
J.A. London, R.G. Wehby/ Brain Research 666 (1994) 270-274
were confined to the anterior tongue by a plastic dam. Tactile stimuli were applied by lightly stroking or tapping a paint brush across the taste field. Thermal stimuli (either 40°C or 10°C distilled water) were applied in the same manner as the sapid stimuli. The following data taking paradigm was employed: 2-60 s of baseline activity, followed by 1-60 s of stimulus application, followed by 120 s of recovery. In some experiments, distilled water was applied for 50-60 s prior to stimulus application. A neuron was considered to have an inhibitory response if, upon application of the stimulus, the activity decreased by 2 standard deviations from baseline activity. In order to recover the location of the activity in the cortex, horseradish peroxidase (HRP) (Sigma) was iontophoresed at locations where taste activity was recorded (3-7 s, 200 ms pulse, 50% duty cycle, 2/xA). At the end of the experiment, the animals were anesthetized and perfused. Their brains were removed and placed in a 30% sucrose buffer solution. Frozen sections were cut at 40 /xm on a sliding microtome. The H R P was visualized by the Hanker-Yates method [3] and counterstained with thionin. Reaction product spots were typically < 100/xm in diameter. Slides were examined with a light microscope (Leitz), and the reaction products located by visual inspection. Photographs and camera lucida drawings of the spot locations were made. Neurons which were either excited or inhibited in response to application of taste stimuli to the anterior tongue were located throughout the dysgranular/ agranular insular cortex (n = 91 animals). Of the 1,489 taste-responsive neurons identified, 1,147 neurons were excited and 342 neurons were inhibited by application of chemical stimuli. Data describing the excitatory neuronal activity will be presented in another paper (London and Wehby, in preparation). There were at least two classes of neurons inhibited by application of chemical stimuli to the anterior tongue: (1) single-component-inhibited neurons: neurons inhibited by only one of the component stimuli; and (2) mixture-inhibited neurons: neurons inhibited by the mixture, but not any single component stimulus alone. Single-component-inhibited neurons. There were a substantial number of neurons (n = 295) that were inhibited by application of a single stimulus. The activity of these neurons was not affected by application of the other chemical stimuli, nor by thermal or tactile stimulation. For 59 of these neurons the degree of inhibition observed was greater for single component stimuli than for the mixture stimulus, suggesting an interaction of the component stimuli (data not shown). These neurons fall into three sub-categories. The first such sub-category contains neurons inhibited solely by application of NaC1 (n = 102 neurons) (Fig. 1). In this experiment, the activity of one spontaneously active
27t
NaCl
Sucrose
KCI I_I.I. LI
L.I___I I _ I _ I
[l I ]I l-l-l-l
I__
L._ILI~_U lilt_IlL_
l-l-l---I
II III U-II-III "
50 ~,v I see
Fig. 1. Response of one cortical neuron to application of three component stimuli. This neuron was inhibited by application of NaC1 to the anterior tongue. Up arrowhead marks start of application of stimulus, down arrowhead marks start of water rinse. Stimuli were applied for 3 s.
neuron was recorded and was inhibited by application of NaCl. Weaker concentrations of NaC1 would diminish, but not block spontaneous activity (data not shown). This neuron had a spontaneous firing rate of 6.0 spikes/s. A second sub-category contains neurons inhibited by application of sucrose (n = 92 neurons) (Fig. 2). In the recording shown, two neurons were identified which were inhibited by application of sucrose. The spontaneous firing rate of the neuron with the larger spike was 1.1 spikes/s. The spontaneous firing rate of the neuron with the smaller spike was 4.2 spikes/s. The third sub-category identified contains
NaCl
Sucrose
KC1
20 # V
0.5 s e c Fig. 2. Responses of two simultaneously recorded cortical neurons to application of three component stimuli. Both neurons were inhibited by application of sucrose to the anterior tongue. Stimuli were applied for 2.5 s. Symbols are the same as for Fig. 1.
272
J.A. London, R.G. Wehby/Brain Research 666 (1994) 270-274
neurons inhibited solely by application of KC1 (n --- 101 neurons) (Fig. 3). The neuron shown here had a spontaneous firing rate of 14.7 spikes/s. Mixture-inhibited neurons. There were 47 neurons that were inhibited only when the mixture solution was applied, but not when the individual stimuli were applied (Fig. 4). The spontaneous firing rate of the neuron in this figure is 5.9 spikes/s. Inhibitory responses comprise a significant proportion of responses recorded in the gustatory cortex of the hamster. Approximately 23% of the neurons that responded to application of taste stimuli were inhibited by this application (342/1147). This value is in agreement with that reported by several other investigators for neurons in the rat cortex. For example, Yamamoto et al. [12] reported that 27% (29/111) of the neurons affected by application of taste stimuli in the rat dysgranular cortex were inhibited. Ogawa et al. [8], reported that 41.2% (21/51) of the neurons in the granular insular cortex, and 29.2% (7/24) of the neurons in the dysgranular insular cortex were inhibited by either one or two stimuli. One group of investigators has reported a frequency of inhibitory responses for a subset of cortical taste neurons at 70% [7]. However, only 30% of these neurons actually exhibited a significant decrease in firing rate [7]. We conclude that taste responsive neurons in the hamster gustatory cortex have a similar distribution of taste responsive neurons as that of the rat gustatory cortex. Recordings of inhibitory responses were made throughout the course of an experiment, and were intermixed with recordings of excitatory responses. Furthermore, in other experiments utilizing the same preparation and stimulus paradigm, EKG and evoked
NaCI
Sucrose
KCI
50~v I 0.5 s e c Fig. 3. Response of a cortical neuron that is inhibited by application of KCI to the anterior tongue. In this case, activity was almost, but not completely inhibited. Application of other stimuli did not effect the activity of the cell. Stimuli were applied for 1.5 s. Symbols are the same as for Fig. 1.
NaCI
Stlcros ~.
KCI
Mixtur,
50 uV 2 sec Fig. 4. Response of a cortical neuron inhibited by application of mixture solution only. There was negligible effect of application of the three component stimuli. Stimuli were applied for 3 s. Symbols are the same as for Fig. 1.
potential recordings before, during and after stimulus application were indistinguishable (unpublished observations). These observations argue against the possibility that the inhibitory responses result from an arousal-state change of the animal, but reflect neuronal activity that occurs in response to application of taste stimuli. The sharpness of tuning of the inhibitory responses is much greater than seen in the rat cortex. The rat granular and dysgranular gustatory cortices contain neurons inhibited by a single stimulus, as well as neurons inhibited by more than one stimulus [8,12]. This difference may be accounted for by three factors. One factor may be that there is a species difference between the rat and the hamster. The distribution of taste bud fields in the oral cavity differs for rat and hamsters. In addition, the hamster has evolved in a very different ecological niche than the rat. As a consequence, the neuronal response to the same stimuli may be different. A second possibility is that there is a difference in the response properties of neurons from different parts of the gustatory cortex. Our recordings were made in the agranular/dysgranular region, whereas the other investigators recorded responses in the granular/dysgranular region. A final reason why
J.A. London, R.G. Wehby / Brain Research 666 (1994) 270-274 Table 1 Distribution of inhibitory responses Neuron type
Number of neurons
Range of spontaneous activity (spikes/s)
Average firing rate (spikes/s)
NaCI KCI Sucrose Mixture
102 101 92 47
0.42-10.1 0.42-16.7 0.35- 9.8 0.44-12.5
7.0 + 0.23 7.3 + 0.36 5.2-t-0.14 6.8 _+0.43
the neurons in the agranular region may be more tightly tuned, and potentially the most interesting reason, is that stimulation in this report was confined to the anterior tongue while other investigators have used whole mouth stimulation. It is possible that excitatory a n d / o r inhibitory inputs from other peripheral taste fields, such as the soft palate or glossopharyngeal fields, converge on taste responsive neurons in the agranul a r / d y s g r a n u l a r cortex, in order to blend taste information from the different peripheral taste fields. This 'mixed input' may serve to sharpen the ability of the animal to detect different stimulus types. Differences between the spontaneous activity of sucrose-inhibited neurons and the other neuron types was recorded (Table 1). The range of spontaneous activity recorded for all categories of the inhibitory response neurons was 0.35-16.7 spikes/s. The average spontaneous firing rates for all the inhibitory response neurons was 5.2 + 0.14 to 7.0 + 0.23 spikes/s. Comparisons of the mean spontaneous firing rates for neurons inhibited by application of NaC1, KC1 or the mixture stimulus (ranging from 6.8 + 0.43 to 7.0 + 0.23 spikes/s) were not significantly different (NaC1 compared to KC1, z = 0.21; NaC1 compared to mixture, z = 0.13, KCI compared to mixture, z = 0.26). However, the mean spontaneous firing rate of sucrose-inhibited neurons (5.2 + 0.14 s p i k e s / s ) was significantly different when compared to the m e a n spontaneous firing rate for all the other neurons in the three cell types (z = 3.29, P < 0.001) with the NaCl-inhibited neurons exhibiting the biggest difference (z = 3.2, P < 0.001). This difference in spontaneous activity of the sucrose-inhibited neurons from the other neurons suggests the possibility of a division, based on neuronal response properties, of cortical activity into hedonic and nonhedonic streams of information. All neurons inhibited by sapid stimuli were located in the d y s g r a n u l a r / a g r a n u l a r insular cortex. While there did not seem to be a strict chemotopic organization in which neurons of one type were restricted to a particular region of the cortex, there did seem to be clusters of like-responsive neurons; a cluster consisted of two to three like-responsive neurons located within 50 /~m of each other (Fig. 2 for example). In 18 animals, clusters of neurons inhibited by application of
273
NaCI were recorded. Often the activities of these neurons were recorded simultaneously. In animals in which two to four clusters were recorded in the same animal (n = 10 animals), clusters were located 100-200 /xm apart. Similar to the neurons that were inhibited by NaCI application, two to three clusters containing two to three sucrose-inhibited neurons were recorded in the same animal (n = 9 animals). These clusters were also located 100-200 /~m apart. Comparable clusters containing two to three KCl-inhibited neurons were also recorded at 1 0 0 - 2 0 0 / x m intervals (n = 9 animals). While component specific neurons appeared in groups, mixture-inhibited neurons were located throughout the agranular insular cortex and did not appear to be clustered within a penetration, or adjacent penetrations. They also did not appear adjacent to a particular class of single-component-inhibited neuron. Our results indicate that the processing of gustatory information is more complex than had recently been thought. There are several types of inhibitory neurons in the gustatory cortex of the hamster. Single-component inhibited-neurons appear clustered together in this cortical region. The role these inhibitory responses may play in the processing of taste information remains to be determined. Some of this work has appeared in abstract form [10]. This work was supported by P50DC00168 and 5T32DC00025. The authors would like to thank Drs. Marion Frank and Joel Zeiger for their critical comments on the manuscript and Mr. Yi-Long Sun for some of the earlier work. [1] Erickson, R.P., Nontraumatic headholders for mammals, Physiol. Behac., 1 (1966) 97-98. [2] Funakoshi, M. and Ninomiya, Y., Relations between the spontaneous firing rate and taste responsiveness of the dog cortical neurons, Brain Res., 262 (1983) 155-159. [3] Hanker, J.S., Yates, P.E., Metz, C.B. and Rustioni A., A new specific, sensitive and non-carcinogenic reagent for the demonstration of horseradish peroxidase, Histochem. J., 9 (1977) 789792. [4] Kosar, E., Grill, H.J. and Norgren, R., Gustatory cortex in the rat. I. Physiological properties and cytoarchitecture, Brain Res., 379 (1986) 329-341. [5] Kosar, E., Grill, H.J. and Norgren, R., Gustatory cortex in the rat. II. Thalamocortical projections, Brain Res., 379 (1986) 342352. [6] Kosar, E. and Schwartz, G.J., Cortical unit responses to chemical stimulation of the oral cavity in the rat, Brain Res., 513 (1990) 212-224. [7] McPheeters, M., Hettinger, T.P.~ Nuding, S.C., Savoy, L.D., Whitehead, M.C. and Frank, M.E., Taste-responsive neurons and their locations in the solitary nucleus of the hamster, Neuroscience, 34 (19990) 745-758. [8] Ogawa, H., Murayama, N. and Hasegawa, K., Difference in receptive field features of taste neurons in rat agranular and dysgranular insular cortices, Exp. Brain Res., 91 (1992) 408-414. [9] Scott, T.R., Plata-Salaman, C.R., Smith, V.L. and Giza, B.K., Gustatory neural coding in the monkey cortex: stimulus intensity, J. Physiol., 65 (1991) 76-86.
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[10] Wehby, R.G. and London, J.A., Inhibition of neuronal responses in the hamster gustatory cortex, Chem. Senses, 17 (1992) 716. [11] Yamamoto, T. and Kitamura, R., A search for the cortical gustatory area in the hamster, Brain Res., 510 (1990) 309-320. [12] Yamamoto, T., Yuyama, N., Kato, T. and Kawamura, Y., Gusta-
tory responses of cortical neurons in rats. I. Response characteristics, J. Neurophysiol., 51 (1984) 616-635. [13] Yamamoto, T., Yuyama, N., Kato, T. and Kawamura, Y., Gustatory responses of cortical neurons in rat. II. Information processing of taste quality, Z Neurophysiol., 53 (1985) 1356-1369.