Visual Neuroscience (1992), 9, 581-593. Printed in the USA. Copyright © 1992 Cambridge University Press 0952-5238/92 $5.00 + .00
Visual responsiveness and direction selectivity of cells in area 18 during local reversible inactivation of area 17 in cats
C. CASANOVA,1 Y. MICHAUD,2 C. MORIN,2 P.A. McKINLEY,1 AND S. MOLOTCHNIKOFF2 'School of Physical and Occupational Therapy, McGill University, Montreal, Canada Departement de Sciences Biologiques et Centre de Recherche en Sciences Neurologiques, Universite de Montreal, Canada
2
(RECEIVED December 17, 1991; ACCEPTED May 20,
1992)
Abstract
We have investigated the effects of inactivation of localized sites in area 17 on the visual responses of cells in visuotopically corresponding regions of area 18. Experiments were performed on adult normal cats. The striate cortex was inactivated by the injection of nanoliters of lidocaine hydrochloride or of 7-aminobutyric acid (GABA) dissolved in a staining solution. Responses of the simple and complex cells of area 18 to optimally oriented light and dark bars moving in the two directions of motion were recorded before, during, and after the drug injection. Two main effects are described. First, for a substantial number of cells, the drug injection provoked an overall reduction of the cell's visual responses. This nonspecific effect largely predominated in the complex cell family (76% of the units affected). This effect is consistent with the presence of long-range excitatory connections in the visual cortex. Second, the inactivation of area 17 could affect specific receptive-field properties of cells in area 18. The main specific effect was a loss of direction selectivity of a number of cells in area 18, mainly in the simple family (more than 53% of the units affected). The change in direction selectivity comes either from a disinhibitory effect in the nonpreferred direction or from a reduction of response in the preferred direction. It is proposed that the disinhibitory effects were mediated by inhibitory interneurones within area 18. In a very few cases, the change of directional preference was associated with a modification of the cell's response profile. These results showed that the signals from area 17 are necessary to drive a number of units in area 18, and that area 17 can contribute to, or at least modulate, the receptive-field properties of a large number of cells in the parastriate area. Keywords: Area 17, Area 18, Cortical inactivation, Cortico-cortical connections, Feedforward connections
et al., 1975; Benevento & Rezak, 1976; Ogren & Hendrickson, 1977; Curcio & Harting, 1978; Harvey, 1980; Dreher et al., 1980; Benevento & Yoshida, 1981; Bullier & Kennedy, 1983; Raczkowski & Rosenquist, 1983; Abramson & Chalupa, 1985). The existence of parallel pathways does not rule out serial processing of the visual signals within the visual cortex, as there are abundant feedforward or ascending projections between the identified visual cortical areas (Wilson, 1968; Weller & Kaas, 1983; Bullier et al., 1984; Symonds & Rosenquist, 1984o,6,; Rockland & Virga, 1990). For instance, anatomical studies have clearly shown that a substantial number of cells in the cat's striate cortex, mainly located in layers II and III, project to area 18 (e.g. Symonds & Rosenquist, 1984ft). These projections are distributed in clusters (Gilbert, 1983, 1985; Price & Blakemore, 1985; Ferrer et al., 1988), and are generally believed to be topographically organized (Mpntero, 1981; Swadlow, 1983). There is however some indication that retinotopically noncorresponding regions of the visual cortex may be connected (Gilbert & Wiesel, 1979). Despite the anatomical evidence of
Introduction Since the early work of Hubel and Wiesel (1962, 1965) from which they proposed a hierarchical relationship between the visual cortical areas, there have been numerous electrophysiological and anatomical studies in cats and more recently in monkeys indicating that visual information is also processed by parallel pathways (see reviews: Stone et al., 1979; Dreher, 1986). It is now well established in both species that thalamic nuclei such as the lateral geniculate nucleus (LGN) and the lateral posterior nucleus-pulvinar (LP-P) complex do project directly to various extrastriate visual areas (Stone & Dreher, 1973; Tretter
Reprint request to: S. Molotchnikoff, Departement de Sciences Biologiques, Universite de Montreal, C.P. 6128, Succ. A, Montreal, Canada, H3C 3J7. Present address of C. Casanova: Department of SurgeryOphthalmology and Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada, J1H 5N4.
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582 feedforward connections between areas 17 and 18, the functional significance of these projections in cat is still unclear (Gilbert, 1985; Ferrer et al., 1988). Acute or chronic lesions of the entire striate cortex have failed to affect in a substantial way specific properties of area 18 cells (Dreher & Cottee, 1975; Donaldson & Nash, 1975). Subsequently, it was reported by Sherk (1978) that the main and only consistent effect of cooling large regions of area 17 was a general reduction of the cell's visual responsiveness. For a number of cells, the reduction was more apparent when slowly rather than fast moving visual stimuli were applied. Although these studies have provided useful information, it should be pointed out that the invasive methods used to inactivate area 17 can potentially lead to various problems noted by the authors themselves. For instance, ablations of area 17 can directly damage area 18 or its blood supply (Sherk, 1978) and cooling probes inactivate a fairly large portion of area 17 and affect all layers of cortex. In addition, none of these studies made a clear distinction between simple and complex-like cells. We have reexamined the influence of the cat's striate cortex on the visual responses of cells in area 18 by using a noninvasive technique of reversible inactivation that has proven to be successful, reliable, and nondamaging in previous studies of cellular connectivity (Casanova & Molotchnikoff, 1990; Molotchnikoff & Hubert, 1990; Casanova et al., 1991ft). Responses to optimally oriented moving light and dark bars of the simple and complex cells of area 18 have been studied prior to and after the chemical blockade of restricted topographically corresponding regions of area 17. Some of these findings have been presented elsewhere in abstract form (Michaud et al., 1989; Casanova et al., 1991a). Methods Animal preparation Experiments were performed with normal adult cats (2.5-3 kg). Animals were premedicated with acepromazine and atropine (1.0 and 0.2 mg/kg, respectively). General anesthesia was induced by intramuscular injection of Ketamine (MTC Pharmaceuticals; 30-40 mg/kg). Lidocaine hydrochloride was applied to surgical wounds and pressure points. After cannulation of the cephalic vein, a tracheotomy was performed and the animal was placed in a stereotaxic frame. A heating pad maintained the core temperature at 37.5°C. The animal was paralyzed by injection of gallamine triethiodide (Flaxedil, Rhone-Poulenc; 10 mg/kg/h) and then artificially ventilated with a gas mixture of N 2 O-O 2 (70:30). The end tidal CO 2 partial pressure was monitored by a capnometer (Hewlett-Packard) and kept between 28 and 35 mm Hg by adjusting the rate and stroke volume of the respiratory pump. The electrocardiogram was monitored throughout the experiment and Fluothane (Ayerst; 0.5%) was added to the gas mixture when needed to maintain an adequate degree of anesthesia. The animal was continually infused with a solution of 5% dextrose in Iactated Ringer's solution and gallamine triethiodide. Pupils were dilated with atropine and nictitating membranes were retracted with local application of neosynephrine. The eyes were protected with contact lenses with no artificial pupils to prevent the cornea from drying. A craniotomy was performed over areas 17 and 18, the dura was reflected, and the electrodes were positioned in visuotopically
C. Casanova el al. corresponding regions (Tusa et al., 1978, 1979). The exposed cortex was covered with warm agar over which wax was melted. Single-unit recordings and visual stimulations Tungsten-in-glass microelectrodes were used to record singleunit activity in area 18 (Levick, 1972). The signals were amplified, displayed on an oscilloscope, and played through an audio monitor. The signals were also passed through a window discriminator and then fed to a computer for peristimulus-time histogram (PSTH) acquisition. Manually controlled stimuli were projected on a translucent screen facing the animal and on which the position of the optic disks were plotted. Bars of variable width and length were used to characterize the receptive-field properties of the isolated unit. Cells were classified as simple or complex according to established criteria (Hubel & Wiesel, 1962). For the quantitative analysis, responses to light and dark moving bars (full screen length, 1-3 deg width, 80% contrast) presented on a CRT located at 57 cm from the eyes were recorded at the optimal orientation and velocity (generally between 8 and 20 deg/s) previously determined. Responses in both directions of movement were recorded in order to evaluate the degree of direction selectivity of the cells. The axis of movement was orthogonal to the optimal orientation. Direction selectivity was quantified as follows: Direction Index (DI) = 1 -
Response in the nonpreferred direction Response in the preferred direction
The number of stimulus presentations was generally ten but could be up to 20, according to the responsiveness of the cell. All stimulations were monocular, and consequently we did not test the effect of the inactivation on the cell's ocular dominance (note that Dreher & Cottee [1975] reported a change in the number of cells that required binocular stimulation only when the contralateral area 17 was ablated). For most binocular cells, only the dominant eye was stimulated. Some cells were virtually unresponsive to monocular stimulation of either eye and were therefore tested binocularly. Injecting protocol The injecting-recording microelectrode consisted of a pulled glass micropipette (tip aperture 15-25 ^m) filled with lidocaine hydrochloride (Xylocaine, Astra; 2(7o) or with GABA (Aldrich; 0.1 mM) dissolved in a saline solution (0.9%). These solutions also contained Chicago sky blue (2%). The electrode was inserted in the head of a nanoliter pump (WPI) that was modified to allow simultaneous recordings (Casanova et al., 1991ft). The rate of injection could be varied from 0-100 nl/min. Control experiments indicated that for a total injection of 120 nl the lidocaine occupies a relatively spheric tissue volume of about 400-500 jtm and that responses within the central 200 /tm are totally abolished while some activity remains in the more peripheral regions. A similar injection of isotonic NaCl failed to modify cortical activity (Casanova & Molotchnikoff, 1990). In the present study, we generally injected around 500 nl of the blocking agent during each test and the spread of these injec-
Responses in area 18 after inactivation of area 17
583
tions was greater than 1 mm. As multiple injections could potentially damage the cells in area 17, no more than three injections were made at a given site. The nature and the proportion of the effects reported below were similar whether lidocaine or GABA was used as the inactivating agent (x 2 = 3.7; P = 0.44). Therefore, no further distinction will be made with respect to GABA and lidocaine in this study.
CAUDAL
Control recordings Successive recordings made before each injection indicated that there was very little variability of the visual responses of the cells tested. We used the total number of spikes/bin generated per trial (with subtraction of spontaneous discharges) to quantify the responses evoked in the forward and reverse direction of movement. The overall mean variation between the control recordings was only 17 ± 12%. Any change resulting from the inactivation was considered significant if it was greater than 40%. The striate cortex was inactivated by injection of the blocking agent at an initial rate of 80-100 nl/min. The injection rate was reduced and maintained throughout the test recording period at 20-40 nl/min when no visual response could be evoked in area 17. Once inactivation of striate cortex was confirmed, responses to light and dark bars of the cell in area 18 were reexamined. Multiple recordings were made during the inactivation to assure the constancy of the responses over time (see Fig. 9). Visual responses were once again characterized after recovery of area 17, which generally occurred 30-40 min after drug injection ceased. For each cell, a blocked/control (B/C) index was calculated by dividing the response during inactivation of area 17 by the control responses recorded prior to the blocking (Malpeli, 1983; Mignard & Malpeli, 1991). Responses were quantified by calculating the total number of spikes/bin in the discharges evoked in the two directions of movement. Indexes of 0 and 1 would indicate, respectively, that responses were totally abolished or not affected. Any enhancement of the discharges would yield an index greater than 1. We also computed a recovery/control (R/C) index to determine the extent to which cells recovered from the disruption of the input from area 17 and also to rule out any prolonged change of the cell's activity caused by an uncontrolled variation in responsiveness. We considered a cell to be affected when its B/C index was less than 0.6 or greater than 1.4, and when the R/C index was between 0.6 and 1.4 (with a few exceptions such as when the unit was lost during the early period of recovery). Histology At the end of each experiment, the animal was sacrificed with an intravenous overdose of Nembutal (Abbot Labs.). The brain was removed from the skull and fixed in a solution of buffered formalin (10%). Frozen serial sections of the visual cortex 60100 ftm in thickness were cut in the frontal plane. Sections confined to the recording sites (area 18) were stained with cresyl violet to locate electrolytic lesions made along the path of the electrode penetrations. Sections of the striate cortex were photographed before and after being stained with neutral red. These sections were used to verify the location and spread of the stained solution of lidocaine or GABA (see Fig. 1). Note that the injecting and the recording electrodes were always separated
D
u
ROSTRAL Fig. 1. Schematic representation of series of coronal sections of the visual cortex showing the lateromedial and rostrocaudal spread of an injection in area 17. Alternate 100-fim sections are presented. Regions highly and moderately stained are represented by filled and dotted areas, respectively. In this particular experiment, three injections were made at three different sites spaced 150 fim apart. The total amount injected was 810 nl. Note that the blocking agent was mainly located in the superficial layers of the cortex. The area 17-18 border is identified by dashed lines. Scale bar = 1 mm.
by several millimeters (in general not less than 4 mm) and histological examination showed that all injections were confined to area 17. There were never any traces of the stained solution near or at the recording site, nor in any part of area 18. Results Inactivation of area 17 The striate cortex was inactivated by the injection of a stained solution of lidocaine or GABA. Fig. 1 shows the lateromedial and rostrocaudal spread of the solution at an injection site. In most cases, injections were restricted to the upper layers of cortex and, for a single injection, the blocking agent rarely extended more than 500-600 /tin from the tip of the injecting pipette. In all experiments, the injection was confined to area 17. During
C. Casanova et al.
584 the experiments, the success of the inactivation was confirmed by the disappearance of the multi-unit responses evoked by the stimulation of area 17 neurons by visual stimuli presented in the appropriate part of the visual field (Fig. 2). Recordings in area 18 General observations A total of 98 cells were recorded from area 18. Out of these, 86 units were completely tested, i.e. their responses to optimally oriented bars were successfully recorded before, during, and after the inactivation of area 17. The cells were classified according to the spatial arrangement of their ON and OFF regions as revealed by their responses to stationary bars (Hubel & Wiesel, 1962) or moving light and dark bars (Kulikowski et al., 1981; Camarda et al., 1985). A total of 42 units out of 86 were classified as simple cells; their receptive fields were characterized by spatially separated ON and OFF subregions. The remaining cells (44 out of 86) were identified as complex cells and their receptive fields consisted of overlapping ON and OFF regions. Simple cells Visual responses of almost three-quarters of the simple cells analyzed (30 out of 42, 71%) were modified by the reversible inactivation of the striate cortex. Effects have been classified in two categories, specific or nonspecific. Nonspecific effects. We observed that the inactivation of area 17 yielded a substantial change of responsiveness of 14 out of 30 (46.7%) units affected in area 18. In most cases (11 cells, 79%), the changes consisted of a decrease of the cell's responsiveness. Only three (21%) simple cells were more visually
CONT.
B
IN ACT. 17
Fig. 2. Example of multi-unit signals recorded from the injectingrecording microelectrode in area 17. The upper trace shows the multiunit response evoked by the visual stimulation (horizontal bars) of area 17 neurons before injection. During the drug injection, no responses could be evoked from the same receptive field. Note also that the overall background activity is markedly reduced. CONT: Control; and INACT: inactivation. Calibration bar = 10 s.
responsive after the injection (Table 1). In both cases, all components of the responses to moving light and dark bars in the forward and reverse directions were equally modified. These general changes have been classified as nonspecific. A representative example is shown in Fig. 3. The cell responded equally for the forward and reverse directions of a moving light bar. Comparable responses were recorded after the reversal of the bar's polarity. It can be seen that the drug inactivation of area 17 provoked a general reduction of the cell's responsiveness, irrespective of the response components and of the bar's contrast. The blockade/control ratios ranged from 0.07-0.6 (14 units, mean of 0.4), which suggests that the overall visual responsiveness of some simple cells in the peristriate area depends largely, and sometimes totally, on inputs from area 17. The distribution of the indices is shown in Fig. 4A. As mentioned above, injection also provoked facilitated effects, but those were rather infrequent. The blockade/control indices of the nonspecific enhancement of the visual responses noted for three units ranged from 1.69-2.16 (mean of 1.9). Specific effects. For the remaining 16 units affected by the chemical blockade of the striate cortex, there was a significant change in the cell's direction selectivity (Fig. 5). In all but three cases, the cells became less selective to the direction of the stimulus motion. The Dh ranged between 0.29 and 1 (mean of 0.67 ± 0.22) before and between -0.14 and 0.63 (mean of 0.24 ± 0.26) during inactivation of area 17. The loss of direction selectivity resulted with about equal frequency from (1) a reduction of excitation in the preferred direction only, (2) an enhancement of the response in the nonpreferred direction only, and (3) a simultaneous reduction and enhancement of the responses in the preferred and nonpreferred directions. The increase of directionality noted for three units resulted from a reduction of the cell's firing rate in the nonpreferred direction. DIs ranged from 0.07-0.49 (mean of 0.22 ± 0.23) prior to and from 0.7-0.84 (mean of 0.76 ± 0.07) during the chemical blockade. A first example is shown in Fig. 6. Prior to the lidocaine injection, the cell showed a clear preference for the downward direction of a moving dark bar (DI = 0.78). Injection of the blocking agent provoked a profound reduction of the response in the preferred direction (DI = -0.14). This type of behavior, i.e. a reduction of excitation in the preferred direction, was observed for five other simple cells. In the second example (Fig. 7), the decrease of direction selectivity resulted from an enhancement of the response in the nonpreferred direction rather than a reduction of the discharges in the preferred direction as illustrated above. The simple cell shown in Fig. 7 responded almost exclusively to the forward direction of movement (DI — 0.96). There was a clear loss of directionality (DI = 0.27) during the drug injection, caused by an enhancement of the response in the nonpreferred direction. Finally, the responses in the preferred as well as in the nonpreferred direction of four units were simultaneously modified. An example is shown in Fig. 8. During the injection, the second discharge evoked by a light bar in the preferred direction (marked by a filled arrow) disappeared while the response in the nonpreferred direction increased. The result of this dual effect was a reduction of direction selectivity (DI = 0.83 and 0.03 before and during injection, respectively). A similar reduction was observed after the contrast reversal of the bar (DI = 0.76 and
Responses in area 18 after inactivation of area 17
585
CONT.
1
I In. INACT.17
2
1
5 _LL REC.
JLj 0.26 prior to and after injection, respectively). Note also that the response profile of the cell was altered. The insert at the bottom of Fig. 8 shows in schematic form the cell's response to the light and dark bars. It is clear that, in addition to the loss of direction selectivity, the envelope of the receptive field was changed when the feedforward connections were temporarily disrupted. An additional example is shown in Fig. 9. The simple cell's response to light and dark bars moving in the forward direction was characterized by five principal peaks or modes. During lidocaine injection, only three modes could be distinguished. In addition to the narrowing of the RF, the amplitude of the last peak evoked by the motion of the light bar was enhanced and the cell became more selective to the direction of the light bar. Note that the response strength and profile during the inactivation were extremely stable between recordings (compare traces 3-4 and traces 9-10) and that a second injection made after area 17 recovered yielded similar changes (traces 6 and 12). This last observation indicates that the effect described did not come from a random fluctuation of the cell's excitability. Changes of response profile were observed for three cells. These results suggest that the striate cortex may participate in the organization of the subregions of the receptive field of some simple cells in area 18. In summary, responses of the majority of the simple cells tested (30 out of 42) were changed by the inactivation of area 17. For almost half of these cells, the effects were nonspecific and mainly consist in a reduction of responses. For the remain-
J
Fig. 3. Overall decrease of the visual responsiveness of a simple cell in area 18 during the inactivation of area 17 (700 nl). Note that all components of the responses evoked by a light or a dark bar are equally affected (nonspecific effect). B/C and R/C ratios are 0.43 and 0.79 for light bar, and 0.16 and 0.76 for dark bar, respectively. CONT: control; INACT: inactivation; and REC: recovery. Bin width = 8 ms. Number of stimulus presentations = 10. Calibration: 1 s, 5 spikes.
ing units, the blockade altered specific properties of the receptive field such as a reduction of the direction selectivity and, in very few cases, a modification of the response profile to moving light and dark bars. These results are summarized in Table 1. Complex cells Twenty-five of the 44 complex cells successfully tested were affected by inactivation of area 17. This proportion is less than that reported above for simple cells. Again, the effects could be classified as nonspecific or specific, but, in contrast to simple cells, nonspecific changes predominated. Nonspecific effects. GABA or lidocaine inactivation provoked a nonspecific change of the responsiveness of most complex cells (19 out of 25 units, 16%). As we reported for simple cells, the effects mainly consisted of a partial or sometimes total abolition of the visual responses (13 units out of 19, 68%). The blocked/control ratios ranged in magnitude from 0.06-0.59 (mean of 0.33; see Fig. 4). A representative example is shown in Fig. 10. The inactivation of area 17 provoked an almost complete suppression of the responses of the cell in both directions of motion of the light and dark bars. It is known that complex cells are insensitive to contrast polarity and it may first seem unnecessary to stimulate these with bars of opposite contrast. We did so to reveal any possible reorganization of the subregions of the receptive field such as the disappearance of an ON or an
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A
Complex Cells
Simple Cells
S i m p l e cells
10 -.
3
1
•
_
Visual responsiveness and direction selectivity of cells in area 18 during local reversible inactivation of area 17 in cats
C. CASANOVA,1 Y. MICHAUD,2 C. MORIN,2 P.A. McKINLEY,1 AND S. MOLOTCHNIKOFF2 'School of Physical and Occupational Therapy, McGill University, Montreal, Canada Departement de Sciences Biologiques et Centre de Recherche en Sciences Neurologiques, Universite de Montreal, Canada
2
(RECEIVED December 17, 1991; ACCEPTED May 20,
1992)
Abstract
We have investigated the effects of inactivation of localized sites in area 17 on the visual responses of cells in visuotopically corresponding regions of area 18. Experiments were performed on adult normal cats. The striate cortex was inactivated by the injection of nanoliters of lidocaine hydrochloride or of 7-aminobutyric acid (GABA) dissolved in a staining solution. Responses of the simple and complex cells of area 18 to optimally oriented light and dark bars moving in the two directions of motion were recorded before, during, and after the drug injection. Two main effects are described. First, for a substantial number of cells, the drug injection provoked an overall reduction of the cell's visual responses. This nonspecific effect largely predominated in the complex cell family (76% of the units affected). This effect is consistent with the presence of long-range excitatory connections in the visual cortex. Second, the inactivation of area 17 could affect specific receptive-field properties of cells in area 18. The main specific effect was a loss of direction selectivity of a number of cells in area 18, mainly in the simple family (more than 53% of the units affected). The change in direction selectivity comes either from a disinhibitory effect in the nonpreferred direction or from a reduction of response in the preferred direction. It is proposed that the disinhibitory effects were mediated by inhibitory interneurones within area 18. In a very few cases, the change of directional preference was associated with a modification of the cell's response profile. These results showed that the signals from area 17 are necessary to drive a number of units in area 18, and that area 17 can contribute to, or at least modulate, the receptive-field properties of a large number of cells in the parastriate area. Keywords: Area 17, Area 18, Cortical inactivation, Cortico-cortical connections, Feedforward connections
et al., 1975; Benevento & Rezak, 1976; Ogren & Hendrickson, 1977; Curcio & Harting, 1978; Harvey, 1980; Dreher et al., 1980; Benevento & Yoshida, 1981; Bullier & Kennedy, 1983; Raczkowski & Rosenquist, 1983; Abramson & Chalupa, 1985). The existence of parallel pathways does not rule out serial processing of the visual signals within the visual cortex, as there are abundant feedforward or ascending projections between the identified visual cortical areas (Wilson, 1968; Weller & Kaas, 1983; Bullier et al., 1984; Symonds & Rosenquist, 1984o,6,; Rockland & Virga, 1990). For instance, anatomical studies have clearly shown that a substantial number of cells in the cat's striate cortex, mainly located in layers II and III, project to area 18 (e.g. Symonds & Rosenquist, 1984ft). These projections are distributed in clusters (Gilbert, 1983, 1985; Price & Blakemore, 1985; Ferrer et al., 1988), and are generally believed to be topographically organized (Mpntero, 1981; Swadlow, 1983). There is however some indication that retinotopically noncorresponding regions of the visual cortex may be connected (Gilbert & Wiesel, 1979). Despite the anatomical evidence of
Introduction Since the early work of Hubel and Wiesel (1962, 1965) from which they proposed a hierarchical relationship between the visual cortical areas, there have been numerous electrophysiological and anatomical studies in cats and more recently in monkeys indicating that visual information is also processed by parallel pathways (see reviews: Stone et al., 1979; Dreher, 1986). It is now well established in both species that thalamic nuclei such as the lateral geniculate nucleus (LGN) and the lateral posterior nucleus-pulvinar (LP-P) complex do project directly to various extrastriate visual areas (Stone & Dreher, 1973; Tretter
Reprint request to: S. Molotchnikoff, Departement de Sciences Biologiques, Universite de Montreal, C.P. 6128, Succ. A, Montreal, Canada, H3C 3J7. Present address of C. Casanova: Department of SurgeryOphthalmology and Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada, J1H 5N4.
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582 feedforward connections between areas 17 and 18, the functional significance of these projections in cat is still unclear (Gilbert, 1985; Ferrer et al., 1988). Acute or chronic lesions of the entire striate cortex have failed to affect in a substantial way specific properties of area 18 cells (Dreher & Cottee, 1975; Donaldson & Nash, 1975). Subsequently, it was reported by Sherk (1978) that the main and only consistent effect of cooling large regions of area 17 was a general reduction of the cell's visual responsiveness. For a number of cells, the reduction was more apparent when slowly rather than fast moving visual stimuli were applied. Although these studies have provided useful information, it should be pointed out that the invasive methods used to inactivate area 17 can potentially lead to various problems noted by the authors themselves. For instance, ablations of area 17 can directly damage area 18 or its blood supply (Sherk, 1978) and cooling probes inactivate a fairly large portion of area 17 and affect all layers of cortex. In addition, none of these studies made a clear distinction between simple and complex-like cells. We have reexamined the influence of the cat's striate cortex on the visual responses of cells in area 18 by using a noninvasive technique of reversible inactivation that has proven to be successful, reliable, and nondamaging in previous studies of cellular connectivity (Casanova & Molotchnikoff, 1990; Molotchnikoff & Hubert, 1990; Casanova et al., 1991ft). Responses to optimally oriented moving light and dark bars of the simple and complex cells of area 18 have been studied prior to and after the chemical blockade of restricted topographically corresponding regions of area 17. Some of these findings have been presented elsewhere in abstract form (Michaud et al., 1989; Casanova et al., 1991a). Methods Animal preparation Experiments were performed with normal adult cats (2.5-3 kg). Animals were premedicated with acepromazine and atropine (1.0 and 0.2 mg/kg, respectively). General anesthesia was induced by intramuscular injection of Ketamine (MTC Pharmaceuticals; 30-40 mg/kg). Lidocaine hydrochloride was applied to surgical wounds and pressure points. After cannulation of the cephalic vein, a tracheotomy was performed and the animal was placed in a stereotaxic frame. A heating pad maintained the core temperature at 37.5°C. The animal was paralyzed by injection of gallamine triethiodide (Flaxedil, Rhone-Poulenc; 10 mg/kg/h) and then artificially ventilated with a gas mixture of N 2 O-O 2 (70:30). The end tidal CO 2 partial pressure was monitored by a capnometer (Hewlett-Packard) and kept between 28 and 35 mm Hg by adjusting the rate and stroke volume of the respiratory pump. The electrocardiogram was monitored throughout the experiment and Fluothane (Ayerst; 0.5%) was added to the gas mixture when needed to maintain an adequate degree of anesthesia. The animal was continually infused with a solution of 5% dextrose in Iactated Ringer's solution and gallamine triethiodide. Pupils were dilated with atropine and nictitating membranes were retracted with local application of neosynephrine. The eyes were protected with contact lenses with no artificial pupils to prevent the cornea from drying. A craniotomy was performed over areas 17 and 18, the dura was reflected, and the electrodes were positioned in visuotopically
C. Casanova el al. corresponding regions (Tusa et al., 1978, 1979). The exposed cortex was covered with warm agar over which wax was melted. Single-unit recordings and visual stimulations Tungsten-in-glass microelectrodes were used to record singleunit activity in area 18 (Levick, 1972). The signals were amplified, displayed on an oscilloscope, and played through an audio monitor. The signals were also passed through a window discriminator and then fed to a computer for peristimulus-time histogram (PSTH) acquisition. Manually controlled stimuli were projected on a translucent screen facing the animal and on which the position of the optic disks were plotted. Bars of variable width and length were used to characterize the receptive-field properties of the isolated unit. Cells were classified as simple or complex according to established criteria (Hubel & Wiesel, 1962). For the quantitative analysis, responses to light and dark moving bars (full screen length, 1-3 deg width, 80% contrast) presented on a CRT located at 57 cm from the eyes were recorded at the optimal orientation and velocity (generally between 8 and 20 deg/s) previously determined. Responses in both directions of movement were recorded in order to evaluate the degree of direction selectivity of the cells. The axis of movement was orthogonal to the optimal orientation. Direction selectivity was quantified as follows: Direction Index (DI) = 1 -
Response in the nonpreferred direction Response in the preferred direction
The number of stimulus presentations was generally ten but could be up to 20, according to the responsiveness of the cell. All stimulations were monocular, and consequently we did not test the effect of the inactivation on the cell's ocular dominance (note that Dreher & Cottee [1975] reported a change in the number of cells that required binocular stimulation only when the contralateral area 17 was ablated). For most binocular cells, only the dominant eye was stimulated. Some cells were virtually unresponsive to monocular stimulation of either eye and were therefore tested binocularly. Injecting protocol The injecting-recording microelectrode consisted of a pulled glass micropipette (tip aperture 15-25 ^m) filled with lidocaine hydrochloride (Xylocaine, Astra; 2(7o) or with GABA (Aldrich; 0.1 mM) dissolved in a saline solution (0.9%). These solutions also contained Chicago sky blue (2%). The electrode was inserted in the head of a nanoliter pump (WPI) that was modified to allow simultaneous recordings (Casanova et al., 1991ft). The rate of injection could be varied from 0-100 nl/min. Control experiments indicated that for a total injection of 120 nl the lidocaine occupies a relatively spheric tissue volume of about 400-500 jtm and that responses within the central 200 /tm are totally abolished while some activity remains in the more peripheral regions. A similar injection of isotonic NaCl failed to modify cortical activity (Casanova & Molotchnikoff, 1990). In the present study, we generally injected around 500 nl of the blocking agent during each test and the spread of these injec-
Responses in area 18 after inactivation of area 17
583
tions was greater than 1 mm. As multiple injections could potentially damage the cells in area 17, no more than three injections were made at a given site. The nature and the proportion of the effects reported below were similar whether lidocaine or GABA was used as the inactivating agent (x 2 = 3.7; P = 0.44). Therefore, no further distinction will be made with respect to GABA and lidocaine in this study.
CAUDAL
Control recordings Successive recordings made before each injection indicated that there was very little variability of the visual responses of the cells tested. We used the total number of spikes/bin generated per trial (with subtraction of spontaneous discharges) to quantify the responses evoked in the forward and reverse direction of movement. The overall mean variation between the control recordings was only 17 ± 12%. Any change resulting from the inactivation was considered significant if it was greater than 40%. The striate cortex was inactivated by injection of the blocking agent at an initial rate of 80-100 nl/min. The injection rate was reduced and maintained throughout the test recording period at 20-40 nl/min when no visual response could be evoked in area 17. Once inactivation of striate cortex was confirmed, responses to light and dark bars of the cell in area 18 were reexamined. Multiple recordings were made during the inactivation to assure the constancy of the responses over time (see Fig. 9). Visual responses were once again characterized after recovery of area 17, which generally occurred 30-40 min after drug injection ceased. For each cell, a blocked/control (B/C) index was calculated by dividing the response during inactivation of area 17 by the control responses recorded prior to the blocking (Malpeli, 1983; Mignard & Malpeli, 1991). Responses were quantified by calculating the total number of spikes/bin in the discharges evoked in the two directions of movement. Indexes of 0 and 1 would indicate, respectively, that responses were totally abolished or not affected. Any enhancement of the discharges would yield an index greater than 1. We also computed a recovery/control (R/C) index to determine the extent to which cells recovered from the disruption of the input from area 17 and also to rule out any prolonged change of the cell's activity caused by an uncontrolled variation in responsiveness. We considered a cell to be affected when its B/C index was less than 0.6 or greater than 1.4, and when the R/C index was between 0.6 and 1.4 (with a few exceptions such as when the unit was lost during the early period of recovery). Histology At the end of each experiment, the animal was sacrificed with an intravenous overdose of Nembutal (Abbot Labs.). The brain was removed from the skull and fixed in a solution of buffered formalin (10%). Frozen serial sections of the visual cortex 60100 ftm in thickness were cut in the frontal plane. Sections confined to the recording sites (area 18) were stained with cresyl violet to locate electrolytic lesions made along the path of the electrode penetrations. Sections of the striate cortex were photographed before and after being stained with neutral red. These sections were used to verify the location and spread of the stained solution of lidocaine or GABA (see Fig. 1). Note that the injecting and the recording electrodes were always separated
D
u
ROSTRAL Fig. 1. Schematic representation of series of coronal sections of the visual cortex showing the lateromedial and rostrocaudal spread of an injection in area 17. Alternate 100-fim sections are presented. Regions highly and moderately stained are represented by filled and dotted areas, respectively. In this particular experiment, three injections were made at three different sites spaced 150 fim apart. The total amount injected was 810 nl. Note that the blocking agent was mainly located in the superficial layers of the cortex. The area 17-18 border is identified by dashed lines. Scale bar = 1 mm.
by several millimeters (in general not less than 4 mm) and histological examination showed that all injections were confined to area 17. There were never any traces of the stained solution near or at the recording site, nor in any part of area 18. Results Inactivation of area 17 The striate cortex was inactivated by the injection of a stained solution of lidocaine or GABA. Fig. 1 shows the lateromedial and rostrocaudal spread of the solution at an injection site. In most cases, injections were restricted to the upper layers of cortex and, for a single injection, the blocking agent rarely extended more than 500-600 /tin from the tip of the injecting pipette. In all experiments, the injection was confined to area 17. During
C. Casanova et al.
584 the experiments, the success of the inactivation was confirmed by the disappearance of the multi-unit responses evoked by the stimulation of area 17 neurons by visual stimuli presented in the appropriate part of the visual field (Fig. 2). Recordings in area 18 General observations A total of 98 cells were recorded from area 18. Out of these, 86 units were completely tested, i.e. their responses to optimally oriented bars were successfully recorded before, during, and after the inactivation of area 17. The cells were classified according to the spatial arrangement of their ON and OFF regions as revealed by their responses to stationary bars (Hubel & Wiesel, 1962) or moving light and dark bars (Kulikowski et al., 1981; Camarda et al., 1985). A total of 42 units out of 86 were classified as simple cells; their receptive fields were characterized by spatially separated ON and OFF subregions. The remaining cells (44 out of 86) were identified as complex cells and their receptive fields consisted of overlapping ON and OFF regions. Simple cells Visual responses of almost three-quarters of the simple cells analyzed (30 out of 42, 71%) were modified by the reversible inactivation of the striate cortex. Effects have been classified in two categories, specific or nonspecific. Nonspecific effects. We observed that the inactivation of area 17 yielded a substantial change of responsiveness of 14 out of 30 (46.7%) units affected in area 18. In most cases (11 cells, 79%), the changes consisted of a decrease of the cell's responsiveness. Only three (21%) simple cells were more visually
CONT.
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IN ACT. 17
Fig. 2. Example of multi-unit signals recorded from the injectingrecording microelectrode in area 17. The upper trace shows the multiunit response evoked by the visual stimulation (horizontal bars) of area 17 neurons before injection. During the drug injection, no responses could be evoked from the same receptive field. Note also that the overall background activity is markedly reduced. CONT: Control; and INACT: inactivation. Calibration bar = 10 s.
responsive after the injection (Table 1). In both cases, all components of the responses to moving light and dark bars in the forward and reverse directions were equally modified. These general changes have been classified as nonspecific. A representative example is shown in Fig. 3. The cell responded equally for the forward and reverse directions of a moving light bar. Comparable responses were recorded after the reversal of the bar's polarity. It can be seen that the drug inactivation of area 17 provoked a general reduction of the cell's responsiveness, irrespective of the response components and of the bar's contrast. The blockade/control ratios ranged from 0.07-0.6 (14 units, mean of 0.4), which suggests that the overall visual responsiveness of some simple cells in the peristriate area depends largely, and sometimes totally, on inputs from area 17. The distribution of the indices is shown in Fig. 4A. As mentioned above, injection also provoked facilitated effects, but those were rather infrequent. The blockade/control indices of the nonspecific enhancement of the visual responses noted for three units ranged from 1.69-2.16 (mean of 1.9). Specific effects. For the remaining 16 units affected by the chemical blockade of the striate cortex, there was a significant change in the cell's direction selectivity (Fig. 5). In all but three cases, the cells became less selective to the direction of the stimulus motion. The Dh ranged between 0.29 and 1 (mean of 0.67 ± 0.22) before and between -0.14 and 0.63 (mean of 0.24 ± 0.26) during inactivation of area 17. The loss of direction selectivity resulted with about equal frequency from (1) a reduction of excitation in the preferred direction only, (2) an enhancement of the response in the nonpreferred direction only, and (3) a simultaneous reduction and enhancement of the responses in the preferred and nonpreferred directions. The increase of directionality noted for three units resulted from a reduction of the cell's firing rate in the nonpreferred direction. DIs ranged from 0.07-0.49 (mean of 0.22 ± 0.23) prior to and from 0.7-0.84 (mean of 0.76 ± 0.07) during the chemical blockade. A first example is shown in Fig. 6. Prior to the lidocaine injection, the cell showed a clear preference for the downward direction of a moving dark bar (DI = 0.78). Injection of the blocking agent provoked a profound reduction of the response in the preferred direction (DI = -0.14). This type of behavior, i.e. a reduction of excitation in the preferred direction, was observed for five other simple cells. In the second example (Fig. 7), the decrease of direction selectivity resulted from an enhancement of the response in the nonpreferred direction rather than a reduction of the discharges in the preferred direction as illustrated above. The simple cell shown in Fig. 7 responded almost exclusively to the forward direction of movement (DI — 0.96). There was a clear loss of directionality (DI = 0.27) during the drug injection, caused by an enhancement of the response in the nonpreferred direction. Finally, the responses in the preferred as well as in the nonpreferred direction of four units were simultaneously modified. An example is shown in Fig. 8. During the injection, the second discharge evoked by a light bar in the preferred direction (marked by a filled arrow) disappeared while the response in the nonpreferred direction increased. The result of this dual effect was a reduction of direction selectivity (DI = 0.83 and 0.03 before and during injection, respectively). A similar reduction was observed after the contrast reversal of the bar (DI = 0.76 and
Responses in area 18 after inactivation of area 17
585
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1
I In. INACT.17
2
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5 _LL REC.
JLj 0.26 prior to and after injection, respectively). Note also that the response profile of the cell was altered. The insert at the bottom of Fig. 8 shows in schematic form the cell's response to the light and dark bars. It is clear that, in addition to the loss of direction selectivity, the envelope of the receptive field was changed when the feedforward connections were temporarily disrupted. An additional example is shown in Fig. 9. The simple cell's response to light and dark bars moving in the forward direction was characterized by five principal peaks or modes. During lidocaine injection, only three modes could be distinguished. In addition to the narrowing of the RF, the amplitude of the last peak evoked by the motion of the light bar was enhanced and the cell became more selective to the direction of the light bar. Note that the response strength and profile during the inactivation were extremely stable between recordings (compare traces 3-4 and traces 9-10) and that a second injection made after area 17 recovered yielded similar changes (traces 6 and 12). This last observation indicates that the effect described did not come from a random fluctuation of the cell's excitability. Changes of response profile were observed for three cells. These results suggest that the striate cortex may participate in the organization of the subregions of the receptive field of some simple cells in area 18. In summary, responses of the majority of the simple cells tested (30 out of 42) were changed by the inactivation of area 17. For almost half of these cells, the effects were nonspecific and mainly consist in a reduction of responses. For the remain-
J
Fig. 3. Overall decrease of the visual responsiveness of a simple cell in area 18 during the inactivation of area 17 (700 nl). Note that all components of the responses evoked by a light or a dark bar are equally affected (nonspecific effect). B/C and R/C ratios are 0.43 and 0.79 for light bar, and 0.16 and 0.76 for dark bar, respectively. CONT: control; INACT: inactivation; and REC: recovery. Bin width = 8 ms. Number of stimulus presentations = 10. Calibration: 1 s, 5 spikes.
ing units, the blockade altered specific properties of the receptive field such as a reduction of the direction selectivity and, in very few cases, a modification of the response profile to moving light and dark bars. These results are summarized in Table 1. Complex cells Twenty-five of the 44 complex cells successfully tested were affected by inactivation of area 17. This proportion is less than that reported above for simple cells. Again, the effects could be classified as nonspecific or specific, but, in contrast to simple cells, nonspecific changes predominated. Nonspecific effects. GABA or lidocaine inactivation provoked a nonspecific change of the responsiveness of most complex cells (19 out of 25 units, 16%). As we reported for simple cells, the effects mainly consisted of a partial or sometimes total abolition of the visual responses (13 units out of 19, 68%). The blocked/control ratios ranged in magnitude from 0.06-0.59 (mean of 0.33; see Fig. 4). A representative example is shown in Fig. 10. The inactivation of area 17 provoked an almost complete suppression of the responses of the cell in both directions of motion of the light and dark bars. It is known that complex cells are insensitive to contrast polarity and it may first seem unnecessary to stimulate these with bars of opposite contrast. We did so to reveal any possible reorganization of the subregions of the receptive field such as the disappearance of an ON or an
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