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Caudal cuneate nucleus projection to the direct thalamic relay to the motor cortex: an electrophysiological study
Brain Research, 360 (1985) 361-365 Elsevier
361
BRE 21247
Caudal cuneate nucleus projection to the direct thalamic relay to the motor cortex: an electrophysiological study ROBERT S. WATERS, YASUHIKO TAMAI* and HIROSHI ASANUMA
The Rockefeller University, New York, NY10021 (U.S.A.) (Accepted August 6th, 1985)
Key words: caudal cuneate nucleus - - ventrolateral nucleus - - ventroposterolateral nucleus - - motor cortex - dorsal column nucleus - - cat - - lemniscal pathway
We previously reported that injection of horseradish peroxidase (HRP) into a physiologically identified region of the thalamus between the ventrolateral nucleus and ventroposterolateral nucleus, VL-VPL, in cat results in labeled cell bodies in the caudal cuneate nucleus (CCN) of the dorsal column nuclei (DCN). We recognized, however, that the spread of HRP to localized regions of less than 1.0 mm distance from the injection site and subsequent uptake by neighboring fibers might have accounted for the resulting label in CCN. In the present study, therefore, we reexamined the DCN input to VL-VPL using a more sensitive physiological method. First, we used the microstimulation technique and corroborated the previous result. In 5 additional preparations, a modified collision procedure was used to ascertain that the same VL-VPL neuron which projects to the motor cortex also receives input from CCN. We report, for the first time, evidence of a lemniscal input to neurons in VL-VPL which are physiologically identified as projecting to the motor cortex. W e previously r e p o r t e d n that injection of horseradish peroxidase ( H R P ) into a physiologically identified region of the thalamus b e t w e e n the ventrolateral nucleus (VL) and v e n t r o p o s t e r o l a t e r a l nucleus (VPL), V L - V P L , in cat results in labeled cell bodies in the caudal cuneate nucleus (CCN) of the dorsal column nuclei (DCN). The spread of H R P a r o u n d the recording electrode was always localized to less than 1.0 mm, as d e t e r m i n e d in unstained tissue; yet we could not rule out the possibility that enzyme uptake occurred in terminals o t h e r than those making contact on V I , - V P L neurons projecting to m o t o r cortex, thus producing a false-positive result. It is well d o c u m e n t e d that cells in V L - V P L receive afferent input from a n u m b e r of different ascending somatosensory regions including spinothalamic tract6, lateral cervical nucleus 5, nucleus 2 7,9 and DCN 4,10, although the latter finding is not without reservation 12. The results from several physiological experiments have d e m o n s t r a t e d that section of the dorsal columns reduces peripheral input to the m o t o r cortex in cats I
and monkeys2, s suggesting that thalamic relay neurons receive input from D C N in both species. Therefore our previous finding was consistent with the hypothesis of a D C N input to V L - V P L neurons projecting to m o t o r cortex although the study was not conclusive. The study did, however, lay the groundwork for the present investigation by allowing us to identify a region in D C N where sensory information may gain access to thalamic neurons in V L - V P L which relay sensory input directly to m o t o r cortex. The present study was u n d e r t a k e n to c o r r o b o r a t e the previous result n by making use of the more sensitive microstimulation m e t h o d to activate neurons in CCN from localized sites within V L - V P L that were physiologically identified as projecting to m o t o r cortex. In 5 additional experiments we used a modified collision p r o c e d u r e to d e m o n s t r a t e that the same V L - V P L neuron which projects to m o t o r cortex also receives input from CCN. The physiological properties of neurons at each effective site in m o t o r cortex, V L - V P L and CCN were also examined.
* On leave of absence from the Department of Physiology, Wakayama Medical College, Wakayama 640, Japan. Correspondence: R.S. Waters. Present address: Department of Anatomy, University of Tennessee, Center for the Health Sciences, 875 Monroe Avenue, Memphis, TN 38163, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
362 Experiments were performed on 9 cats weighing between 2.5 and 3.5 kg. In the first part of the study 4 cats were prepared using inhalation anesthesia composed of a mixture of 40% oxygen and 60% nitrous oxide supplemented with 1.0-2.0% halothane. The animal's head was placed in a stereotaxic apparatus, the bone overlying the motor cortex was removed and a Lucite chamber was attached to the skull with dental impression wax. A second recording chamber was placed stereotaxically such that an electrode passing through the center was directed to the V L - V P L border region. The caudal portion of the cerebellum was removed by gentle aspiration to expose the brainstem in the vicinity of the obex region. The dura overlying the caudal cuneate nucleus contralateral to the recording chamber was removed and the skin was retracted around the region to form a pool over the exposed brainstem and cerebellum; the pool was filled with warm mineral oil. All wound tissues were infiltrated with a long-lasting local anesthetic (Zyljectin, Abbott). The animal's head was rigidly held by attaching a head holding device to several stainless steel screws implanted into the skull. The head holding device was anchored to a baseplate. The gas anesthesia was then discontinued and the animal maintained on low doses of ketamine (12 mg/kg, Parke-Davis) for the remainder of the experiment. Through the anterior chamber an array of 7 tungsten-in-glass microelectrodes was inserted into the bank of the cruciate sulcus and fixed at a depth of 1.2 mm below the surface. Through the caudal chamber a single tungsten-in-glass microelectrode was driven into the thalamus. An additional microelectrode was inserted in the CCN by a micromanipulator mounted on the baseplate. Single cathodal pulses (0.2 ms duration; 30 ~A, maximum intensity) were delivered simultaneously through the motor cortex electrodes while a single electrode was driven into V L - V P L . Whenever an activated neuron was encountered, the responsible motor cortex stimulating electrode was noted and threshold intensity examined. The response latencies at threshold and 2x threshold were measured, the neuron's ability to follow rapid repeated stimulation (> 100 Hz) was examined~ and in those cases when the neuron was spontaneously active the collision test was used. The activated neuron was examined for responsiveness to cu-
taneous stimulation (bending the hair and touching the skin with a camel hair brush), deep muscle stimulation and joint rotation. When the thalamic neuron was identified as antidromically activated and had a clear receptive field, the recording electrode was fixed into that location and used to deliver single pulse microstimulation, while a single tungsten-inglass microelectrode was inserted into the caudal cuneate nucleus located lateral to the obex region. Several penetrations were made in CCN while stimulating VL-VPL. When activated CCN neurons satisfied the same criteria for antidromic activation, receptive field measurements were made as described above. The receptive field properties were then compared to receptive fields of neurons around the effective stimulating electrodes in motor cortex and VL-VPL. In 5 experiments, a modified collision procedure was used to ascertain whether the same V L - V P L neuron projecting to motor cortex also received short-latency input from CCN. The experimental paradigm was identical to that described above with the exceptions that cats were anesthetized with Nembutal (35 mg/kg) during surgery and supplemented with Nembutal throughout the experiment and all surgery and recording were accomplished while animals remained in the stereotaxic apparatus. Using this procedure, microstimulation was delivered to electrodes in motor cortex and CCN while recording from V L - V P L . AntidromicaUy activated responses recorded from V L - V P L neurons could be made to collide with orthodromically activated responses of the same cells using appropriate intervals of stimulus delay from each stimulating site. Seventy-five electrode penetrations were made in the thalamus and 85 neurons were identified as antidromically activated from motor cortex. Of this number 39% had clear receptive fields and were located within the border between VL and VPL. The majority of these neurons were activated from the rostral lateral portion of motor cortex with a median intensity threshold of 18~A (range 12-30#A) and a median latency of 1.2 ms (range 0.9-1.9 ms). In addition, 36 penetrations were made into CCN and 23 neurons which had clear receptive fields were activated antidromically. The median threshold intensity for activation of CCN neurons from V L - V P L was 11 /~A (range 3-30 ~tA), but in each cat at least one CCN
363 neuron was antidromically activated with thalamic stimulation of 10/~A or less and the experiments were carried out utilizing these low-threshold neurons. The median latency for activation was 1.4 ms and ranged between 1.0 and 2.0 ms. A typical experiment is illustrated in Fig. 1. A schematic representation of the general stimulating and recording procedure is shown in Fig. 1A. In this particular example, stimulation (single pulse 0.2 ms cathodal followed by 0.1 ms anodal; 30/~A maximum intensity) delivered to an electrode located in the rostral part of the motor cortex activated a neuron in V L - V P L . This neuron was identified as being antidromically activated on the basis of the collision test and was activated with a threshold intensity of 25 /~A with a latency of 0.9 ms. A photograph taken from several superimposed oscilloscope sweeps is shown in Fig. lB. Following identification of the antidromically activated thalamic neuron, the recording
electrode was fixed at this location and used for stimulation while a third electrode was inserted in CCN in the vicinity of the obex region to record activated neurons following V L - V P L stimulation. Altogether two electrode penetrations (approximately 1 mm apart) were made in this trial, and field potentials following thalamic stimulation were recorded during each penetration. Antidromically activated neurons were encountered in only one of these penetrations. A photograph of several oscilloscope superimposed sweeps showing the response of the CCN neurons is shown in Fig. 1B on the right. This CCN neuron was activated with a threshold current of less than 20/~A and had a response latency of 1.4 ms following stimulation of V L - V P L . Marking lesions were made by passing anodal current of 10/~A for 30 s through the relevant electrodes. Histological reconstruction of the sites of these neurons, indicated by an asterisk, in V L - V P L (center)
x x
X
20 ,leA
B
COR
Fig. 1. General stimulating and recording paradigm employed in tracing dorsal column nuclei (DCN) input to motor cortex. A: schematic representation of stimulating and recording technique used in motor cortex (MCx), thalamic relay nucleus (VL/VPL) and caudal cuneate nucleus (CCN) of the DCN. B: oscilloscope tracing of antidromically activated neurons recorded at VL-VPL (left) and CCN (right). The response latencies and thresholds are also indicated. Time scale, 1.0 ms/division. C: microprojection drawings of histological reconstruction of location of l~sion (asterisk) at effective sites in MCx (left), VL-VPL (center) and CCN (right). CUR, cruciate sulcus; COR, coronal sulcus; VA, ventroanterior nucleus; VL, ventrolateral complex; LP, lateral posterior complex; VPM, ventroposteromedial nucleus; VPL, ventroposterolateral nucleus; F'I'C, central tegmental field; IC, internal capsule; ZI, zona incerta; OT, optic tract; SNR, substantia nigra; ION, inferior olivary nucleus; FTL, lateral tegmental field.
364 and CCN (right) are shown in Fig. 1C. The location of the effective electrode in motor cortex is shown at the left in Fig. 1C. The receptive fields of these antidromically activated neurons in V L - V P L and CCN were examined using natural peripheral stimulation as already described and both of these were activated by light touch to the dorsal wrist. This receptive field overlapped with the receptive field recorded from neurons around the motor cortex stimulating electrode. The results from the microstimulation study provide further evidence for a direct sensory relay from DCN to motor cortex via the V L / V P L thalamic border region, but these results are still subject to the same type of criticism as raised for the H R P experiment outlined in the previous paper ll, namely spread of stimulating current (although restricted to less than t 0 0 # m in the best cases) might still activate neurons in the neighboring VPL region and thus produce a false-positive result. To eliminate the issue of current spread in V L - V P L we used a modified collision technique and recorded from the electrode in V L - V P L while stimulating both motor cortex and CCN. The value of the present study lay in setting the stage for this next step in the investigation. Once the electrodes were positioned in V L - V P L and CCN using the antidromic identification procedure described above, we then orthodromically activated the V L - V P L neuron from CCN and collided the impulse with the antidromic spike generated by stimulation of motor cortex. In addition, stimulation was also delivered to neighboring sites around the activated CCN neurons. In most examined cases, CCN stimulation yielded a compound response; however, in 6 cases we were able to orthodromically activate single thalamic neurons. Evidence for a D C N input to a thalamic neuron projecting directly to the motor cortex was judged on the basis of whether the CCN-activated thalamic neuronal response would collide with the antidromically activated response of the same neuron following stimulation of the motor cortex. A n example of the results from one activated thalamic neuron is illustrated in Fig. 2. In the upper two traces, labeled 'Control', responses recorded from V L - V P L are shown following motor cortex stimulation alone (top trace) and CCN stimulation alone (lower trace). The stimulus artifact for the control responses is indicated by arrows at the beginning of each trace. In this example the V L - V P L neuron was antidromically ac-
tivated from motor cortex with a latency ot 0.9 ms and orthodromically activated from CCN with a response latency of 2.0-2.1 ms. (Stimulation delivered through the electrode in V L - V P L antidromically activated a CCN neuron with a latency of 1.4 ms.) The lower 5 traces show the results of test cases where combined stimulation from motor cortex and CCN and/or motor cortex stimulation alone was used. When motor cortex stimulation was delayed 2.4 ms following CCN stimulation collision occurred and the antidromically activated spike disappeared. This can be seen in the third trace labeled CCN + MCx. 2.4 ms. If, however. CCN stimulation is turned off, the motor cortex-activated spike is then seen at the position of the arrow shown in trace 'a'. The trace to the right, labeled 'b', shows the antidromically acti-
MCx- Control
~lj~ v'~m~q'eev'~'
A
CCN- Control
I
~/ " ~ k ~ ~ i
2.4
mL
J
CCN + MCx 4.0 ms
~ III
-
CCN+ MCx 5,1 m,
b
1.0 MS
Fig. 2. Modified collision technique. In the upper two traces. labeled Control. 4 superimposed oscilloscope tracings are shown for the antidromicaUy activated V L - V P L neuron response following stimulation of motor cortex (MCx, top trace) and for the orthodromically activated V L - V P L neuron response following stimulation of CCN alone (lower trace). In the third trace. 3 superimposed oscilloscope tracings are shown for the combined stimulation of CCN and MCx with a 2.4-ms delay between stimulation. Tracing a shows 3 additional oscilloscope sweeps with CCN stimulation turned off. The photograph b shows superimposed oscilloscope tracings of MCx stimulation alone. The last two photographs show superimposed traces of CCN and MCx stimulation with delay times of 4.0 and 5.1 ms. A time scale (1.0 ms) is shown at lower right.
365 vated V L - V P L neuron response following m o t o r cortex stimulation alone. Note the identical waveshape of the thalamic neuron following both C C N and m o t o r cortex stimulation. This further suggests that the same thalamic n e u r o n was activated from both stimulating sites. In the lower two traces the result of c o m b i n e d CCN and m o t o r cortex is seen with delay times of 4.0 and 5.1 ms, respectively. A t these longer delay times collision does not occur in the thalamic neuron. These results strongly support the view of a lemniscal input to thalamic neurons in the V L - V P L b o r d e r region and these neurons in turn send their axons directly to the m o t o r cortex. While these results in cat provide strong evidence for a D C N contribution to the direct thalamic relay to m o t o r cortex, this issue remains unresolved in monkey. Thalamic neurons in ventroposterolateralis pars oralis (VPLo) in m o n k e y are r e p o r t e d to project to regions of m o t o r cortex having similar receptive fields 2. A b l a t i o n or cooling of sensory cortex did not eliminate the sensory input to m o t o r cortex 3, while section of dorsal columns greatly reduced the afferent input 8. These results strongly suggest that neurons at the b o r d e r region between V L and V P L receive peripheral input through D C N and relay this input to m o t o r cortex in both cats and monkeys. However, Tracey et al. 12 could not find labeled cell bodies in D C N in the m o n k e y following injection of H R P into VPLo. The injections were m a d e into V P L o based on physiological identification of field potentials elicited by p e r i p h e r a l nerve stimulation. In addition, these investigators were unable to find labeled 1 Asanuma, H., Larsen, K.D. and Zarzecki, P., Peripheral input pathways projecting to the motor cortex in the cat, Brain Research, 172 (1979) 197-208. 2 Asanuma, H., Larsen, K.D. and Yumiya, H., Direct sensory pathways to the motor cortex in the monkey: a basis of cortical reflexes. In H. Asanuma and V. Wilson (Eds.), Integration in The Nervous System, Igaku-shoin, Tokyo, 1979, pp. 223-238. 3 Asanuma, H., Larsen, K.D. and Yumiya, H., Peripheral input pathways to the monkey motor cortex, Exp. Brain Res., 38 (1980) 349-355. 4 Berkley, K., Spatial relationships between the terminations of somatic sensory motor pathways in the rostral brainstem of cats and monkeys. II. Cerebellar projections compared with those of the ascending somatic sensory pathways in lateral diencephalon, J. Comp. Neurol., 220 (1983) 220-229. 5 Boivie, J., The termination of the cervicothalamic tract in the cat. An experimental study with silver impregnation methods, Brain Research, 19 (1970) 333-360. 6 Boivie, J., The termination of the spinothalamic tract in the cat. An experimental study with silver impregnation methods, Exp. Brain Res., 12 (1971) 331-353.
fibers in V P L o after injection of tritiated amino acids into D C N . Since the selection of injection sites in V P L o was based on peripheral stimulation the question of whether neurons at the site of injection also project to m o t o r cortex cannot be answered. M o r e recently Berkley examined the input to the b o r d e r region in cats and m o n k e y s and r e p o r t e d degenerating fibers at the V L - V P L b o r d e r in cats and at caudal V P L o in m o n k e y s following ablation of D C N 4. In part, the discrepancy between the early anatomical and physiological studies might arise from the difficulty of delineating V P L o histologically4. It is possible that Tracey et al. 12 injected H R P into the center of V P L o where field potentials were present but projection fibers from D C N are not found. While these findings in m o n k e y are difficult to resolve at this time, our experiments in cat d e m o n s t r a t e that neurons in CCN terminate on thalamic cells in V L - V P L which project directly to m o t o r cortex. Although we have not investigated the question of species differences, we present a m e t h o d which could be used to further explore this question in monkeys. The authors thank Dr. R o b e r t Schor for his helpful comments during this research, Mrs. A n d r e Jean Marie and Mrs. Maryse A u b u r g for technical support, and Mrs. M. W a t e r s for editing. This research was s u p p o r t e d by N I H G r a n t NS-10705 to H . A . and NSF G r a n t BNS 83-17662 to R . S . W . ; Y.T. is a recipient of the International Fellowship from the Japanese Ministry of Education.
7 Boivie, J., Grant, G. and Silfvenius, H., Projection from the nucleus Z to the thalamus of the cat, Acta Physiol. Scand., 80 (1970) l l A . 8 Brinkman, J., Bush, B.M. and Porter, R., Deficient influences of peripheral stimuli on precentral neurones in monkeys with dorsal column lesions, J. Physiol. (London), 276 (1978) 27-48. 9 Grant, G., Boivie, J. and Silfvenius, H., Course and termination of fibres from the nucleus Z of the medulla oblongata. An experimental light microscopical study in the cat, Brain Research, 55 (1973) 55-70. 10 Hand, P.J. and Van Winkle, T., The efferent connections of the feline nucleus cuneatus, J. Comp. Neurol., 171 (1977) 83-110. 11 Tamai, Y., Waters, R.S. and Asanuma, H., Caudal cuneate nucleus projection to the direct thalamic relay to motor cortex in cat: An electrophysiological and anatomical study, Brain Research, 323 (1984) 360-364. 12 Tracey, D.J., Asanuma, C., Jones, E.G. and Porter, R., Thalamic relay to motor cortex: afferent pathways from brain stem, cerebellum, and spinal cord in monkeys, J. Neurophysiol., 44 (1980) 532-554.
361
BRE 21247
Caudal cuneate nucleus projection to the direct thalamic relay to the motor cortex: an electrophysiological study ROBERT S. WATERS, YASUHIKO TAMAI* and HIROSHI ASANUMA
The Rockefeller University, New York, NY10021 (U.S.A.) (Accepted August 6th, 1985)
Key words: caudal cuneate nucleus - - ventrolateral nucleus - - ventroposterolateral nucleus - - motor cortex - dorsal column nucleus - - cat - - lemniscal pathway
We previously reported that injection of horseradish peroxidase (HRP) into a physiologically identified region of the thalamus between the ventrolateral nucleus and ventroposterolateral nucleus, VL-VPL, in cat results in labeled cell bodies in the caudal cuneate nucleus (CCN) of the dorsal column nuclei (DCN). We recognized, however, that the spread of HRP to localized regions of less than 1.0 mm distance from the injection site and subsequent uptake by neighboring fibers might have accounted for the resulting label in CCN. In the present study, therefore, we reexamined the DCN input to VL-VPL using a more sensitive physiological method. First, we used the microstimulation technique and corroborated the previous result. In 5 additional preparations, a modified collision procedure was used to ascertain that the same VL-VPL neuron which projects to the motor cortex also receives input from CCN. We report, for the first time, evidence of a lemniscal input to neurons in VL-VPL which are physiologically identified as projecting to the motor cortex. W e previously r e p o r t e d n that injection of horseradish peroxidase ( H R P ) into a physiologically identified region of the thalamus b e t w e e n the ventrolateral nucleus (VL) and v e n t r o p o s t e r o l a t e r a l nucleus (VPL), V L - V P L , in cat results in labeled cell bodies in the caudal cuneate nucleus (CCN) of the dorsal column nuclei (DCN). The spread of H R P a r o u n d the recording electrode was always localized to less than 1.0 mm, as d e t e r m i n e d in unstained tissue; yet we could not rule out the possibility that enzyme uptake occurred in terminals o t h e r than those making contact on V I , - V P L neurons projecting to m o t o r cortex, thus producing a false-positive result. It is well d o c u m e n t e d that cells in V L - V P L receive afferent input from a n u m b e r of different ascending somatosensory regions including spinothalamic tract6, lateral cervical nucleus 5, nucleus 2 7,9 and DCN 4,10, although the latter finding is not without reservation 12. The results from several physiological experiments have d e m o n s t r a t e d that section of the dorsal columns reduces peripheral input to the m o t o r cortex in cats I
and monkeys2, s suggesting that thalamic relay neurons receive input from D C N in both species. Therefore our previous finding was consistent with the hypothesis of a D C N input to V L - V P L neurons projecting to m o t o r cortex although the study was not conclusive. The study did, however, lay the groundwork for the present investigation by allowing us to identify a region in D C N where sensory information may gain access to thalamic neurons in V L - V P L which relay sensory input directly to m o t o r cortex. The present study was u n d e r t a k e n to c o r r o b o r a t e the previous result n by making use of the more sensitive microstimulation m e t h o d to activate neurons in CCN from localized sites within V L - V P L that were physiologically identified as projecting to m o t o r cortex. In 5 additional experiments we used a modified collision p r o c e d u r e to d e m o n s t r a t e that the same V L - V P L neuron which projects to m o t o r cortex also receives input from CCN. The physiological properties of neurons at each effective site in m o t o r cortex, V L - V P L and CCN were also examined.
* On leave of absence from the Department of Physiology, Wakayama Medical College, Wakayama 640, Japan. Correspondence: R.S. Waters. Present address: Department of Anatomy, University of Tennessee, Center for the Health Sciences, 875 Monroe Avenue, Memphis, TN 38163, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
362 Experiments were performed on 9 cats weighing between 2.5 and 3.5 kg. In the first part of the study 4 cats were prepared using inhalation anesthesia composed of a mixture of 40% oxygen and 60% nitrous oxide supplemented with 1.0-2.0% halothane. The animal's head was placed in a stereotaxic apparatus, the bone overlying the motor cortex was removed and a Lucite chamber was attached to the skull with dental impression wax. A second recording chamber was placed stereotaxically such that an electrode passing through the center was directed to the V L - V P L border region. The caudal portion of the cerebellum was removed by gentle aspiration to expose the brainstem in the vicinity of the obex region. The dura overlying the caudal cuneate nucleus contralateral to the recording chamber was removed and the skin was retracted around the region to form a pool over the exposed brainstem and cerebellum; the pool was filled with warm mineral oil. All wound tissues were infiltrated with a long-lasting local anesthetic (Zyljectin, Abbott). The animal's head was rigidly held by attaching a head holding device to several stainless steel screws implanted into the skull. The head holding device was anchored to a baseplate. The gas anesthesia was then discontinued and the animal maintained on low doses of ketamine (12 mg/kg, Parke-Davis) for the remainder of the experiment. Through the anterior chamber an array of 7 tungsten-in-glass microelectrodes was inserted into the bank of the cruciate sulcus and fixed at a depth of 1.2 mm below the surface. Through the caudal chamber a single tungsten-in-glass microelectrode was driven into the thalamus. An additional microelectrode was inserted in the CCN by a micromanipulator mounted on the baseplate. Single cathodal pulses (0.2 ms duration; 30 ~A, maximum intensity) were delivered simultaneously through the motor cortex electrodes while a single electrode was driven into V L - V P L . Whenever an activated neuron was encountered, the responsible motor cortex stimulating electrode was noted and threshold intensity examined. The response latencies at threshold and 2x threshold were measured, the neuron's ability to follow rapid repeated stimulation (> 100 Hz) was examined~ and in those cases when the neuron was spontaneously active the collision test was used. The activated neuron was examined for responsiveness to cu-
taneous stimulation (bending the hair and touching the skin with a camel hair brush), deep muscle stimulation and joint rotation. When the thalamic neuron was identified as antidromically activated and had a clear receptive field, the recording electrode was fixed into that location and used to deliver single pulse microstimulation, while a single tungsten-inglass microelectrode was inserted into the caudal cuneate nucleus located lateral to the obex region. Several penetrations were made in CCN while stimulating VL-VPL. When activated CCN neurons satisfied the same criteria for antidromic activation, receptive field measurements were made as described above. The receptive field properties were then compared to receptive fields of neurons around the effective stimulating electrodes in motor cortex and VL-VPL. In 5 experiments, a modified collision procedure was used to ascertain whether the same V L - V P L neuron projecting to motor cortex also received short-latency input from CCN. The experimental paradigm was identical to that described above with the exceptions that cats were anesthetized with Nembutal (35 mg/kg) during surgery and supplemented with Nembutal throughout the experiment and all surgery and recording were accomplished while animals remained in the stereotaxic apparatus. Using this procedure, microstimulation was delivered to electrodes in motor cortex and CCN while recording from V L - V P L . AntidromicaUy activated responses recorded from V L - V P L neurons could be made to collide with orthodromically activated responses of the same cells using appropriate intervals of stimulus delay from each stimulating site. Seventy-five electrode penetrations were made in the thalamus and 85 neurons were identified as antidromically activated from motor cortex. Of this number 39% had clear receptive fields and were located within the border between VL and VPL. The majority of these neurons were activated from the rostral lateral portion of motor cortex with a median intensity threshold of 18~A (range 12-30#A) and a median latency of 1.2 ms (range 0.9-1.9 ms). In addition, 36 penetrations were made into CCN and 23 neurons which had clear receptive fields were activated antidromically. The median threshold intensity for activation of CCN neurons from V L - V P L was 11 /~A (range 3-30 ~tA), but in each cat at least one CCN
363 neuron was antidromically activated with thalamic stimulation of 10/~A or less and the experiments were carried out utilizing these low-threshold neurons. The median latency for activation was 1.4 ms and ranged between 1.0 and 2.0 ms. A typical experiment is illustrated in Fig. 1. A schematic representation of the general stimulating and recording procedure is shown in Fig. 1A. In this particular example, stimulation (single pulse 0.2 ms cathodal followed by 0.1 ms anodal; 30/~A maximum intensity) delivered to an electrode located in the rostral part of the motor cortex activated a neuron in V L - V P L . This neuron was identified as being antidromically activated on the basis of the collision test and was activated with a threshold intensity of 25 /~A with a latency of 0.9 ms. A photograph taken from several superimposed oscilloscope sweeps is shown in Fig. lB. Following identification of the antidromically activated thalamic neuron, the recording
electrode was fixed at this location and used for stimulation while a third electrode was inserted in CCN in the vicinity of the obex region to record activated neurons following V L - V P L stimulation. Altogether two electrode penetrations (approximately 1 mm apart) were made in this trial, and field potentials following thalamic stimulation were recorded during each penetration. Antidromically activated neurons were encountered in only one of these penetrations. A photograph of several oscilloscope superimposed sweeps showing the response of the CCN neurons is shown in Fig. 1B on the right. This CCN neuron was activated with a threshold current of less than 20/~A and had a response latency of 1.4 ms following stimulation of V L - V P L . Marking lesions were made by passing anodal current of 10/~A for 30 s through the relevant electrodes. Histological reconstruction of the sites of these neurons, indicated by an asterisk, in V L - V P L (center)
x x
X
20 ,leA
B
COR
Fig. 1. General stimulating and recording paradigm employed in tracing dorsal column nuclei (DCN) input to motor cortex. A: schematic representation of stimulating and recording technique used in motor cortex (MCx), thalamic relay nucleus (VL/VPL) and caudal cuneate nucleus (CCN) of the DCN. B: oscilloscope tracing of antidromically activated neurons recorded at VL-VPL (left) and CCN (right). The response latencies and thresholds are also indicated. Time scale, 1.0 ms/division. C: microprojection drawings of histological reconstruction of location of l~sion (asterisk) at effective sites in MCx (left), VL-VPL (center) and CCN (right). CUR, cruciate sulcus; COR, coronal sulcus; VA, ventroanterior nucleus; VL, ventrolateral complex; LP, lateral posterior complex; VPM, ventroposteromedial nucleus; VPL, ventroposterolateral nucleus; F'I'C, central tegmental field; IC, internal capsule; ZI, zona incerta; OT, optic tract; SNR, substantia nigra; ION, inferior olivary nucleus; FTL, lateral tegmental field.
364 and CCN (right) are shown in Fig. 1C. The location of the effective electrode in motor cortex is shown at the left in Fig. 1C. The receptive fields of these antidromically activated neurons in V L - V P L and CCN were examined using natural peripheral stimulation as already described and both of these were activated by light touch to the dorsal wrist. This receptive field overlapped with the receptive field recorded from neurons around the motor cortex stimulating electrode. The results from the microstimulation study provide further evidence for a direct sensory relay from DCN to motor cortex via the V L / V P L thalamic border region, but these results are still subject to the same type of criticism as raised for the H R P experiment outlined in the previous paper ll, namely spread of stimulating current (although restricted to less than t 0 0 # m in the best cases) might still activate neurons in the neighboring VPL region and thus produce a false-positive result. To eliminate the issue of current spread in V L - V P L we used a modified collision technique and recorded from the electrode in V L - V P L while stimulating both motor cortex and CCN. The value of the present study lay in setting the stage for this next step in the investigation. Once the electrodes were positioned in V L - V P L and CCN using the antidromic identification procedure described above, we then orthodromically activated the V L - V P L neuron from CCN and collided the impulse with the antidromic spike generated by stimulation of motor cortex. In addition, stimulation was also delivered to neighboring sites around the activated CCN neurons. In most examined cases, CCN stimulation yielded a compound response; however, in 6 cases we were able to orthodromically activate single thalamic neurons. Evidence for a D C N input to a thalamic neuron projecting directly to the motor cortex was judged on the basis of whether the CCN-activated thalamic neuronal response would collide with the antidromically activated response of the same neuron following stimulation of the motor cortex. A n example of the results from one activated thalamic neuron is illustrated in Fig. 2. In the upper two traces, labeled 'Control', responses recorded from V L - V P L are shown following motor cortex stimulation alone (top trace) and CCN stimulation alone (lower trace). The stimulus artifact for the control responses is indicated by arrows at the beginning of each trace. In this example the V L - V P L neuron was antidromically ac-
tivated from motor cortex with a latency ot 0.9 ms and orthodromically activated from CCN with a response latency of 2.0-2.1 ms. (Stimulation delivered through the electrode in V L - V P L antidromically activated a CCN neuron with a latency of 1.4 ms.) The lower 5 traces show the results of test cases where combined stimulation from motor cortex and CCN and/or motor cortex stimulation alone was used. When motor cortex stimulation was delayed 2.4 ms following CCN stimulation collision occurred and the antidromically activated spike disappeared. This can be seen in the third trace labeled CCN + MCx. 2.4 ms. If, however. CCN stimulation is turned off, the motor cortex-activated spike is then seen at the position of the arrow shown in trace 'a'. The trace to the right, labeled 'b', shows the antidromically acti-
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CCN- Control
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Fig. 2. Modified collision technique. In the upper two traces. labeled Control. 4 superimposed oscilloscope tracings are shown for the antidromicaUy activated V L - V P L neuron response following stimulation of motor cortex (MCx, top trace) and for the orthodromically activated V L - V P L neuron response following stimulation of CCN alone (lower trace). In the third trace. 3 superimposed oscilloscope tracings are shown for the combined stimulation of CCN and MCx with a 2.4-ms delay between stimulation. Tracing a shows 3 additional oscilloscope sweeps with CCN stimulation turned off. The photograph b shows superimposed oscilloscope tracings of MCx stimulation alone. The last two photographs show superimposed traces of CCN and MCx stimulation with delay times of 4.0 and 5.1 ms. A time scale (1.0 ms) is shown at lower right.
365 vated V L - V P L neuron response following m o t o r cortex stimulation alone. Note the identical waveshape of the thalamic neuron following both C C N and m o t o r cortex stimulation. This further suggests that the same thalamic n e u r o n was activated from both stimulating sites. In the lower two traces the result of c o m b i n e d CCN and m o t o r cortex is seen with delay times of 4.0 and 5.1 ms, respectively. A t these longer delay times collision does not occur in the thalamic neuron. These results strongly support the view of a lemniscal input to thalamic neurons in the V L - V P L b o r d e r region and these neurons in turn send their axons directly to the m o t o r cortex. While these results in cat provide strong evidence for a D C N contribution to the direct thalamic relay to m o t o r cortex, this issue remains unresolved in monkey. Thalamic neurons in ventroposterolateralis pars oralis (VPLo) in m o n k e y are r e p o r t e d to project to regions of m o t o r cortex having similar receptive fields 2. A b l a t i o n or cooling of sensory cortex did not eliminate the sensory input to m o t o r cortex 3, while section of dorsal columns greatly reduced the afferent input 8. These results strongly suggest that neurons at the b o r d e r region between V L and V P L receive peripheral input through D C N and relay this input to m o t o r cortex in both cats and monkeys. However, Tracey et al. 12 could not find labeled cell bodies in D C N in the m o n k e y following injection of H R P into VPLo. The injections were m a d e into V P L o based on physiological identification of field potentials elicited by p e r i p h e r a l nerve stimulation. In addition, these investigators were unable to find labeled 1 Asanuma, H., Larsen, K.D. and Zarzecki, P., Peripheral input pathways projecting to the motor cortex in the cat, Brain Research, 172 (1979) 197-208. 2 Asanuma, H., Larsen, K.D. and Yumiya, H., Direct sensory pathways to the motor cortex in the monkey: a basis of cortical reflexes. In H. Asanuma and V. Wilson (Eds.), Integration in The Nervous System, Igaku-shoin, Tokyo, 1979, pp. 223-238. 3 Asanuma, H., Larsen, K.D. and Yumiya, H., Peripheral input pathways to the monkey motor cortex, Exp. Brain Res., 38 (1980) 349-355. 4 Berkley, K., Spatial relationships between the terminations of somatic sensory motor pathways in the rostral brainstem of cats and monkeys. II. Cerebellar projections compared with those of the ascending somatic sensory pathways in lateral diencephalon, J. Comp. Neurol., 220 (1983) 220-229. 5 Boivie, J., The termination of the cervicothalamic tract in the cat. An experimental study with silver impregnation methods, Brain Research, 19 (1970) 333-360. 6 Boivie, J., The termination of the spinothalamic tract in the cat. An experimental study with silver impregnation methods, Exp. Brain Res., 12 (1971) 331-353.
fibers in V P L o after injection of tritiated amino acids into D C N . Since the selection of injection sites in V P L o was based on peripheral stimulation the question of whether neurons at the site of injection also project to m o t o r cortex cannot be answered. M o r e recently Berkley examined the input to the b o r d e r region in cats and m o n k e y s and r e p o r t e d degenerating fibers at the V L - V P L b o r d e r in cats and at caudal V P L o in m o n k e y s following ablation of D C N 4. In part, the discrepancy between the early anatomical and physiological studies might arise from the difficulty of delineating V P L o histologically4. It is possible that Tracey et al. 12 injected H R P into the center of V P L o where field potentials were present but projection fibers from D C N are not found. While these findings in m o n k e y are difficult to resolve at this time, our experiments in cat d e m o n s t r a t e that neurons in CCN terminate on thalamic cells in V L - V P L which project directly to m o t o r cortex. Although we have not investigated the question of species differences, we present a m e t h o d which could be used to further explore this question in monkeys. The authors thank Dr. R o b e r t Schor for his helpful comments during this research, Mrs. A n d r e Jean Marie and Mrs. Maryse A u b u r g for technical support, and Mrs. M. W a t e r s for editing. This research was s u p p o r t e d by N I H G r a n t NS-10705 to H . A . and NSF G r a n t BNS 83-17662 to R . S . W . ; Y.T. is a recipient of the International Fellowship from the Japanese Ministry of Education.
7 Boivie, J., Grant, G. and Silfvenius, H., Projection from the nucleus Z to the thalamus of the cat, Acta Physiol. Scand., 80 (1970) l l A . 8 Brinkman, J., Bush, B.M. and Porter, R., Deficient influences of peripheral stimuli on precentral neurones in monkeys with dorsal column lesions, J. Physiol. (London), 276 (1978) 27-48. 9 Grant, G., Boivie, J. and Silfvenius, H., Course and termination of fibres from the nucleus Z of the medulla oblongata. An experimental light microscopical study in the cat, Brain Research, 55 (1973) 55-70. 10 Hand, P.J. and Van Winkle, T., The efferent connections of the feline nucleus cuneatus, J. Comp. Neurol., 171 (1977) 83-110. 11 Tamai, Y., Waters, R.S. and Asanuma, H., Caudal cuneate nucleus projection to the direct thalamic relay to motor cortex in cat: An electrophysiological and anatomical study, Brain Research, 323 (1984) 360-364. 12 Tracey, D.J., Asanuma, C., Jones, E.G. and Porter, R., Thalamic relay to motor cortex: afferent pathways from brain stem, cerebellum, and spinal cord in monkeys, J. Neurophysiol., 44 (1980) 532-554.