There has long been a need for a good method of staining neurones from which intracellular recordings have been made. Recently Kravitz et al. 6 and Stretton ...
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Morphological identification of physiologically defined neurones in the cat spinal cord There has long been a need for a good method of staining neurones from which intracellular recordings have been made. Recently Kravitz e t al. 6 and Stretton and Kravitz 8 described a technique consisting of intracellular iontophoretic injection of a fluorescent dye (Procion Yellow M4RS, ICI). The dye was found to stain both somas and fine branches of neurones in the lobster abdominal ganglion revealing their morphology and relations to other cells. We have now found that this technique gives good results with several types of cells in the cat spinal cord. This will be illustrated with the cells of origin of the ventral spinocerebellar tract (VSCT). Glass micropipettes (with broken tips of 1.5-2.0 #m) were filled with a nearly saturated (about 5 %) solution of Procion Yellow. Those with a 15-30 M f ] resistance were found suitable for both staining and recording. They did not block more than K-citrate electrodes of the same size, and before dye injection the cells did not show more signs of damage than seen after impalement with electrodes of other types. During dye injection the duration of spike potentials increased to 2-3 msec as seen from Fig. 2 F - G . Larger amounts of dye damaged the cells as indicated by a decrease of the spike amplitude, block of antidromic invasion and disappearance of membrane potential. The synaptic potentials, both EPSPs and IPSPs, were fairly unchanged also after deterioration of the spike mechanism. G o o d staining of somas and dendrites of the VSCT cells (which are of the same size as a-motoneurones) required passage of about 20 nA for 10 min. The injection of the dye with a constant hyperpolarizing current proved to be as effective as with square pulses, provided that the electrodes were not blocked. Low current intensities (10-30 nA) used for longer times seem to be preferable as the cells deteriorate more slowly under these conditions. The same electrode can be used for staining of several cells. Before fixation of the cord at least 4 h are needed for diffusion of the dye for good staining of dendrites and axons. The best results were obtained when the spinal cords, after perfusion with Ringer solution, were removed and kept for 30-36 h in low temperature (1-4°C) before fixation in 10% neutralized formalin. After de-
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Fig. I. Photomicrographs of soma, dendrites and axon of a VSCT cell. The same cell as in Fig. 2M. A shows a part of the soma and the dendrites seen in one section, with the same enlargement as in Fig. 2M. In B the part of the dendrite, indicated in A by broken line, is shown with higher enlargement. In C and D there are two parts of the axon, 0.2 and 1.4 mm away from the soma. Note weaker staining more distally. The same enlargement as in B. Brain Research, 20 (1970) 323-326
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hydration in ethyl alcohol and embedding in paraffin the spinal cords were cut and the serial sections (15/~m thick) were mounted on slide glasses. Under the fluorescence microscope (using a darkfield condenser and Leitz filters Schott BG 12-3 and K 510 for the exciting and emitted light respectively) the cells stained with Procion Yellow were easily distinguished from other cells. They are bright yellow-orange on a greenish background. Of 35 injected VSCT cells all but 5 were stained. The photomicrographs in Fig. 1 show a part of soma (A), dendrites (B) and axon (C, D) o f a VSCT cell. In Fig. 2 are shown camera lucida drawings from photomicrographs of 2 VSCT cells, one of them (M) the same as in Fig. 1. The drawings were made by superimposing tracings from several consecutive sections. The dendrites of VSCT cells could be traced for about 500 #m from the somas and the axons up to somewhat more than 2 mm. The VSCT cells were identified by antidromic invasion from the contralateral spinal half (at thoracic level) and by their afferent inputl,a, 7. The two cells of Fig. 2 belong to two subgroups of the VSCT cells. The cell in L was monosynaptically excited by group Ib afferents from quadriceps as seen in Fig. 2 A - D . The cell in M belongs to the spinal border cells (SBC) of Cooper and Sherrington ~ which were recently shown by Burke et al. 1 to give origin to VSCT fibres in the cat. It was monosynaptically excited by group Ia afferents from quadriceps (cf. Fig. 2 0 - Q ) . Cells of the latter type were found mostly in the outer parts of the ventral horn but also within the motor nuclei which confirms findings by Burke et al. (to be published). The VSCT cells excited from Ib afferents were found more dorsally and medially, as described by Hubbard and Oscarsson 5, but also in the neighbourhood of the SBC region in the ventral horn. Both types are of similar size and send their axons medially. Whenever it was possible to trace the axons far enough, they crossed the midline via the ventral commissure not more than I00 # m cranially to the somas. Four axons were followed for 200-300/zm in the contralateral ventral funiculus. Several SBCs had many thin and abundantly branching dendrites, some going far out in the white matter. These results are in keeping with conclusions of Ha and Liu 4 concerning VSCT cells and the trajectory of their axons. Fig. 2. Drawings of 2 VSCT cells injected with Procion Yellow and intracellular records from the same ceils. The drawings of each cell were made by superimposingcamera lucida tracings from photomicrographs of 8 (in L) and 11 (in M) consecutive sections. The two cells L and M are shown with smaller enlargement in K together with the axon of the more lateral cell (M). Records in A-J and N-R (upper traces, intracellular potentials; lower traces, cord dorsum potentials) are from the cells in L and M respectively. As shown with double volley technique in A-D the medial cell was excited monosynapticallyfrom group Ib afferents from quadriceps (Q). The figures above records give stimulation strength for the first and second shock in multiples of threshold for the nerve. The cell was also inhibited from the sural nerve (Sur). The series of records in F-J show the antidrmnic spikes evoked by stimulation of the contralateral spinal half (co cord). They were taken before (F), at the beginning (G) and later during injection of the dye (H-J). O-Q show that the monosynaptic EPSP in the other cell was evoked from the group Ia afferents from quadriceps (Q). N shows the antidromic spike and R the IPSP from high threshold muscle afferents from quadriceps. Voltage calibration for intracellular records in A-D is in D, for E is in E, for F-J is in J and for N-R is in R. All time calibrations are 1 + 5 msec, indicated by smaller and bigger bars respectively. The cell in L was stained with a constant current of 25 nA and 10 msec pulses of 30 hA, applied during 25 rain. For the cell in M only a constant current (20 nA during 15 min) was used. Brain Research, 20 (1970) 323-326
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Besides V S C T cells and a - m o t o n e u r o n e s * , smaller cells (e.g. some interneurones) a n d fibres could also be satisfactorily stained with Procion Yellow. O u r findings thus confirm the wide applicability of the technique suggested by Stretton and Kravitz 8. We are i n d e b t e d to Professor P.-I. Brhnemark, L a b o r a t o r y of Experimental Biology, D e p a r t m e n t o f A n a t o m y , U n i v e r s i t y of G 6 t e b o r g , G 6 t e b o r g , for providing facilities for fluorescence microscopy.
Department of Physiology, University of Gi~teborg, Gi~teborg (Sweden)
E. JANKOWSKA S. LINDSTROM
1 BURKE, R., LUNDBERG, A., AND WEIGHT, F., Gower's tract and 'spinal border cells', Acta physiol.
scand., 74 (1968) 16A. 2 COOPER, S., AND SHERRINGTON, C. S., Gower's tract and spinal border cells, Brain, 63 0940) 123-134.
3 ECCLES,J. C., HUBBARD,J. I., ANDOSCARSSON,O., Intracellular recording from cells of the ventral spinocerebellar tract, J. Physiol. (Lond.), 158 (1961) 486-516. 4 HA, H., AND LIU, C.-N., Cell origin of the ventral spinocerebellar tract, J. comp. Neurol., 133 (1968) 185-206. 5 HtraBARD, J. I., AND OSCARSSON,O., Localization of the cell bodies of the ventral spinocerebellar tract in lumbar segments of the cat, J. comp. Neurol., 118 (1962) 199-204. 6 KRAVlTZ,E. A., STgETTON,A. O. W., ALVAREZ,J., ANDFURSHPAN,E. J., Determination of neuronal geometry using an intracellular dye injection technique, Fed. Proc., 27 (1968) 749. 7 LUNDBERG,A., ANDOSCARSSON,O., Functional organization of the ventral spino-cerebellar tract in cat. IV. Identification of units by antidromic activation from the cerebellar cortex, Acta physiol. scand., 54 (1962) 252-269. 8 S~RET'roN,A. O. W., ANDKRAVXTZ,E. A., Neuronal geometry: Determination with a technique of intracellular dye injection, Science, 162 (1968) 132-134. (Accepted March 20th, 1970)
* After this manuscript was submitted for publication a report on staining of motoneurones with Procion Yellow, by Barrett and Graubard, appeared in Brain Research, 18 (1970) 565-568.
Brain Research, 20 (1970) 323-326
