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Mammalian spinal motoneuron dendrites respond with cyclic degeneration and regeneration after ventral root crush. In the following experiments, the cross-sectional dendritic profile of rat lamina IX, medial, motoneurons under the T2 vertebra were analyzed after mincing the dorsal horn (normals, 14, 30, 60, 90 days postoperative (DPO); N = 6 animals/DPO). The spinal cords were impregnated by the tungstate modification of the Golgi technique. Individual lengths along dendritic segments between branching points were measured from coded slides, the data were computerized, and the dendrites were reconstructed by computer. Interanimal statistical comparisons were made by ANOVA a priori and Newman-Keuls test a posteriori. At 14 DPO, there was a statistically significant (P less than 0.05) increase in the number of dendritic segments, dendritic and serpentine length, and number of segments emanating from the soma compared with normal intact rats and with all other postoperative days. At 30 and 60 DPO, these parameters returned to normal values; however, there were many long, unbranched dendrites. At 90 DPO, there was a statistically significant (P less than 0.05) decrease in motoneuron dendritic serpentine length and segments. These data show that partially deafferented rat spinal motoneurons undergo a biphasic response; an initial growth phase followed by a degenerative phase.

Qualitative behavioral deficits after bilateral lesion of the rat dorsal funiculus at T12–13 were studied during prelesion, postoperative (14 to 21 days postlesion), and terminal (the last 4 days before killing at 30, 45, 60, 90, or 120 days postlesion) periods. Utilizing each animal (36 with lesion, 5 sham stress) as its own preoperative control, hind limb performance was rated for five behavioral components (slips, recovery, manner of traverse, movement, tail position) on four apparatuses (wide platform, narrow platform, ladder, parallel bars). Significant postoperative deficits ( N = 36) occurred on slips, recovery, and manner; Spearman coefficients between apparatus pairs suggested that either of the platforms and/or parallel bars could have demonstrated most component deficits. Sham-stress controls ( N = 5) showed no significant deficits in postoperative performance. Postoperatively, lesion size correlated with some performance deficits. Also, significant postoperative deficits were obtained with 25 to 87% damage of the dorsal column, though solely in the manner of traverse as an individual component; additional destruction (90 to 100%) of the dorsal column added several other deficits. Larger dorsal funicular lesions which included at least 50% of the corticospinal tract significantly affected performance still further, particularly on the parallel bars. Terminal performance ( N = 24), in general, was significantly poorer than that at prelesion periods on 23 (of 29 possible) comparisons. Terminal performance degraded considerably at 90 and 120 days compared with post-operative behavior. Because, in related deafferentation studies, changes in bouton renewal and neuronal excitability can occur at these late postlesion periods, eroded behavioral deficits may reflect a loss of synaptic elements critical in mediating stable behavior.

Fetal spinal cord grafts survive for long periods in host spinal cord. We explored the ability of such grafts into a lesion in C3 fasciculus gracilis to influence the expected deterioration of hind limb performance following this lesion. Ten adult Sprague-Dawley rats were placed on a schedule of 23 h off and 1 h on water. These subjects were then trained to traverse a narrow platform for a water reward. Criterion was established as 10 trials/day completed in 15 s a trial for 2 consecutive days. Animals were ranked for hind limb and forelimb performance utilizing slips, recovery, and manner of traversing the platform. In a triple-blind experimental design, the animals' identification was coded and laminectomy performed at C3. The fasciculus gracilis was bilaterally transected at the rostral and caudal borders of the segment and aspirated. Five subjects were chosen at random immediately after the lesion was made for the implantation of two 1-mm segments of E14 spinal cord from fetuses of timed pregnant Sprague-Dawley dams at the time of the lesion. Coded lesion and lesion-transplant animals were then tested after 21, 30, 45, 60, and 90 days until criterion or for 4 days. Hind limb performance of the lesion-only (ANOVA chi 2 = 13.04, df 5; P less than 0.05) and lesion-transplant groups (ANOVA chi 2 = 11.82, df 5; P less than 0.05) showed that the C3 fetal transplants significantly decreased (P less than 0.05) the severity of hind limb deficit at 21 days postlesion and significantly (P less than 0.05) decreased the expected foot slips and limb recovery to the platform that was expected and did develop in lesion-only animals at 90 days. There were no significant differences in forelimb performance (ANOVAs P greater than 0.05). There were few small-caliber nerve fibers in the fasciculus gracilis at the level of C1 indicating that reinnervation was not a major factor in the reduction of the deficit.

There appears to be specificity in the regrowth of nerve fibers in the fish central nervous system. However, the distribution of terminal boutons is unknown. This study was undertaken to ascertain the reinnervation of neurons by boutons in the regenerated goldfish spinal cord. The spinal cord of 60 goldfish was transected and a segment 2 cm caudal to the lesion was examined 5, 10, 15, 20, 25, 30, 45, and 60 days postoperatively. In another group of animals the spinal cord was retransected after 60 days regeneration time and examined 5 days later to show contributions of long regenerated tracts. The spinal cord segment was stained for boutons by the Rasmussen technique. Boutons were counted on the soma of neurons in medial intermediate gray, ventral horn motoneurons, and ventral horn interneurons and in addition, on primary dendrite of motoneurons. Animals were observed daily for the return of normal swimming. Neurons in the intermediate gray region showed a gradual linear decrease in boutons for 30 days and they remained at this level from 30 to 60 days. Motoneuron soma and primary dendrite showed an abrupt decrease in boutons at 5 days, with a linear increase in boutons to normal levels by 60 days. Retransection of the spinal cord after 60 days did not result in bouton loss on soma or dendrite. These data indicate that the motoneuron was originally innervated by descending long tracts but was now reinnervated by other tracts. Ventral-horn interneuron soma showed an initial loss of boutons followed by return to normal numbers by 30 days and hyperinnervation between 30 and 60 days. Retransection resulted in the return of boutons to normal levels. Thus, this interneuron was reinnervated by regenerated descending tracts. Despite this synaptic reorganization of spinal neurons, normal swimming returned. We conclude that specificity of regeneration is not selective for soma or dendrite of spinal neurons but is probably selective for the neuronal pools or circuits necessary for swimming.

Alteration of spinal motoneurons can be induced by axotomy, deafferentation, or deafferentation of spinal interneurons. In our experiment, dendritic profiles of rat lamina IX motoneurons were studied 1.5 to 2.5 mm rostral to a T2, T3 (interface) hemisection [six per group of normal, 7-, 14-, 30-, 60-, and 90-day postoperative (DPO) animals]. The spinal cord was impregnated using the Golgi technique and individual dendritic segments measured from coded slides, the data were computerized, and the length, number of segments, and dendritic profile were reconstructed. These data were heteroscedastic (alpha less than 0.005) making them inappropriate for ANOVA. However, the data did show two size classes of motoneurons at 14, 30, 60, and 90 DPO. At 7 DPO, there was a reduced dendritic field. At 14 to 60 DPO, there were gigantic motoneurons that were many times larger than normal or other neurons in these operated groups. At 90 DPO a large motoneuron was present. These data show that individual motoneurons have different regenerative dendritic responses after spinal injury.

The effect of puromycin on synaptic renewal on spinal neurons 1 to 1.5 mm rostral to the site of a T1-T2 interface after hemisection was studied. Ninety-five Long-Evans rats were utilized (five animals per group) 10, 20, 30, 45, 60, and 90 days posthemisection. The groups consisted of normal animals, those with spinal cord hemisection alone, those with Gelfoam sponge saturated with 0.9% saline placed in the lesion, or those with a Gelfoam sponge saturated with 1.0 m m puromycin dihydrochloride in 0.9% saline placed in the lesion (at surgery). The spinal cords were processed for the light microscopic impregnation of presynaptic boutons. Counts of boutons (lamina IV and IX motoneurons) were made from coded material and analyzed statistically. After spinal cord hemisection alone, there was a cyclic significant renewal of presynaptic boutons on the somata of lamina IV and IX neurons with decreases ( P P P P

The secondary transneuronal effect of bilateral, dorsal column lesion (T12) on the frequency of boutons on cells in the lateral ventroposterolateral nucleus (VPL) was studied. Rats were killed 1, 2, 3, 7, 14, 30, 45, 60, 90, or 120 days postoperative (DPO), and bouton counts (Rasmussen stain) were made on the soma, or along 5- and 10-μm segments of the proximal dendrite branching from soma, in the rostral two-thirds of the VPL. The soma diameter also was measured of those neurons chosen for bouton counts on the circumference of the soma. Significant, increased afferentiation along a 5-μm segment of the proximal dendrite occurred during the first 3 days postlesion compared with longer survival times. Along the 10-μm segment, as at 5 μm on the proximal dendrite, bouton counts at 2 DPO were significantly higher than at 30 and 120 DPO. This rapid change in afferentation during the first week postlesion mirrors similar effects reported in the nucleus gracilis of these same cases. Bouton counts along the 5-μm segment of the proximal dendrite decreased significantly to less than normal at 30 and 120 DPO, indicating the nonstable and protracted character of afferentation after the distal cord lesion. A significant, positive correlation was obtained between boutons on the soma and proximal dendrite (5 μm segment) in cases with lesions, suggesting parallel innervation patterns for these adjacent neuronal components during 120 postoperative days. There was no significant change in the bouton counts or diameter of the soma during postoperative days. These results suggest that lesion of the thoracic dorsal column may result in subsequent rapid bouton patterning on the lateral VPL neurons, presumably mediated by their medial lemniscal afferent fibers. The postlesion source(s) of afferentation remains an intriguing, and as yet unanswered, question.

After peripheral axotomy, mammalian spinal motoneuron dendrites have been shown to cyclically degenerate and regenerate and after spinal dorsal horn mince to initially increase in length and then atrophy with time. In our experiment the dendritic profile of rat lamina IX motoneurons in the spinal segment under the T2 vertebra was studied for 90 postoperative days after bilateral somatomotor cortex ablation. The spinal cords were impregnated by the tungstate modification of the Golgi technique. Individual lengths along dendritic segments between branching points were measured from coded slides, the data computerized, and the dendrites reconstructed by computer. Interanimal statistical comparisons were made a priori (ANOVA) and a posteriori (Neuman-Keuls). There were no statistical differences in dendritic length or in the number of dendritic segments with DPO. However, there were shifts in the internal branching of all dendrites. The lack of statistical significance between control and experimental animals in numbers of dendritic branches with shifting internal dendritic branching was evidence that the motoneuron dendrites were actively growing but internal dendritic branch losses and gains were indistinguishable and approximately equally along the dendrite. These shifts in internal dendritic branching may be due to loss of information from the descending tracts.

Dendritic alteration can be induced by peripheral axotomy of central nervous system neurons. In the following experiment the dendritic profile of rat lamina IX motoneurons in the spinal cord segment under the T2 vertebra after ventral root crush (controls, 14, 30, 60, and 90 days postoperative (DPO), six animals per group) were studied. The spinal cord was impregnated by the Golgi technique, individual dendritic segments were measured from coded slides, the data computerized, and the dendrites reconstructed by computer. The dendritic parameters generated were length of the dendritic segment between branches, total number of branches, number of terminal branches, and serpentine length (total length of entire dendrite). Inter- and intraanimal statistical comparisons were made by ANOVA a priori and the Newman-Keuls test a posteriori. There were statistically significant losses and gains of dendritic branches and numbers of terminal dendrites (lowest, 14 and 60 days; highest, 30 and 90 days). The serpentine length of the dendrites showed the same statistically significant oscillations. There were statistically significant differences in average dendritic parameters (calculated from distance along dendritic segments) after ventral root crush. However, 17% of the neurons at 14 days, 100% at 30 days, 33% at 60 days, and 17% at 90 days differed from control values in serpentine length and at 14 DPO, 50%; 30 DPO, 50%; 60 DPO, 67%; and 90 DPO, 70% of the neurons had numbers of dendritic segments different from control. These data show that there is a cyclic degeneration and regeneration of motoneuron dendrites after ventral root crush for at least 90 postoperative days.

The effect of bilateral dorsal column lesion on the afferentation and diameter of neurons in the nucleus gracilis (NG) of the rat was studied during 120 postoperative days. After bilateral, low thoracic lesion of the dorsal column, the animals were killed 1, 2, 3, 7, 14, 30, 45, 60, 90, or 120 days later, and cells in the rostral, middle, or caudal NG were selected and measured for soma diameter and number of boutons on these same somas. The results showed significant reduction in soma diameter during the first 2 weeks postoperatively compared with normals, and also significant changes (increases or decreases in diameter) in survival times during 120 postoperative days. Soma diameter and number of boutons on the soma were positively correlated at all NG levels and survival times. Extrapolations of those values to total surface area and total surface area of soma covered by boutons, show that (i) neither normal nor deafferented soma are totally innervated, and (ii) normal innervation values are maintained in parallel with fluctuations in the total surface area of the soma. These results emphasize the dynamic changes occurring upon and in the postsynaptic cell after deafferentation, and imply a mechanism which regulates the interaction of quantity of presynaptic afferent fibers upon, and size of, the postsynaptic cell body.

Protein metabolism in brain and spinal cord of rats subjected to spinal cord hemisection or a control operation was examined using the incorporation of [3H]lysine into trichloracetic acid (TCA)-precipitable protein and TCA-soluble fractions as an indicator. Groups of animals were studied 30, 45, 60, and 90 days after a left spinal hemisection or laminectomy alone at T2. One hour prior to utilization, the animals were injected subcutaneously with 200 μCi [3H]lysine. Tissue samples were taken from left and right somatomotor and occipital cortex and in spinal cord as far as 14 mm rostral and caudal to the site of hemisection. In brain, the uptake of [3H]lysine into protein ant TCA-soluble fractions was greater (P spinal hemisection > laminectomy controls). In rats with spinal hemisection of various durations, the average values of protein radioactivity fluctuated significantly (P < 0.05) with time with peaks at 30 and 60 days postoperation. These differences did not appear in control groups with laminectomies. In localized regions of spinal cord, the site of spinal hemisection had the highest (P < 0.05) protein and TCA-soluble radioactivity of all regions examined. Also, the left (lesion) side of the spinal cord had a greater average protein radioactivity than the right side in all groups of animals with spinal hemisection. These data indicate that spinal lesions produce long-term alterations in spinal cord amino acid metabolism both at the site of lesion and in regions not directly traumatized. The cyclic fluctuations of average protein radioactivity in spinal cord may correlate (Spearman rank correlation coefficient, 0.9 to 1.0) with the cyclic renewal of boutons in the rat with spinal hemisection described in earlier studies. This suggests that general metabolic factors may be important in the regenerative processes of the central nervous system.

The learning and retention of conditioned avoidance responses by goldfish were studied in a two-way avoidance task designed to minimize the demands of the task for forebrainless fish. Forebrainless fish were less active than normal fish in both pretraining response rate and between-trial cross rate. Forebrainless fish learned the avoidance response as well as normal fish with a minimal barrier and a 10-sec light-shock interval. Forebrainless fish were inferior to normal fish after insertion of a larger barrier which reduced the area for avoidance, concomitant with a 4-sec light-shock interval. Removal of the telencephalon produced significant deficits in the performance of a previously learned avoidance response; however, there was evidence for partial savings. It is proposed that the normal function of the telencephalon is best considered to be that of facilitating the processing of information.

Although the central nervous system of the mammal is reported not to regenerate, axonal sprouting has recently been demonstrated in various regions of brain and spinal cord. The following study investigates the regenerative capacity of 72 rat spinal cords following left hemisection at T2. In addition to nine normals, rats were killed at 5, 7, 14, 30, 60, and 90 days after making the lesion. Three animals per groups were prepared for Golgi, Protargol-eosin-cresyl violet staining, and electron microscopy. Three additional animals had hemisections at C5 and the cord from C5 to T5 was stained by the Fink-Heimer method. Six animals had hemisections at T2 and the cords were subsequently rehemisected 90 days later at C5; three animals were prepared for the Fink-Heimer stain and three for electron microscopy. The dendrites of the reactive motor neurons proximal to the site of lesion became varicose from day 10 to day 60–90 after the lesion, until the entire dendrite was replete with irregularly shaped varicosities. At 15 days postoperatively, processes grew into the reactive neural zone and by day 30 could be identifified as axons (0.1–0.5 μ in diameter) and dendrites. The axons grew in fascicles, usually free of neuroglial cell processes. The amjority of the axons formed axodendritic synapses on the indentations of the dendritic varicosities by 60–90 days postoperatively. The Fink-Heimer stain reveled that a limited number of axons regenerated from long tracts into the reactive neural zone. Most regenerated axons appear to be axonal sprouts from the operated and unoporated portions of the spinal cord. The central nervous system of the rat does regenerate, but the regenerating axons do not grow past the reactive neural zone and thus do not reach the neuroglial scar.

The effect of forebrain ablation on brightness discrimination in goldfish has been studied. Cardiac deceleration, a conditioned autonomic response, was used as the measure of discrimination in a series of goldfish. Normal and forebrainablated fish were trained to discriminate between two gray stimuli of different brightness. If the experimental animal made this brightness discrimination in thirty-five trials it was subsequently tested on a black and white stimulus pair to see whether the fish was capable of stimulus generalization. If the fish did not make the brightness discrimination, it was trained to discriminate a different set of stimuli to show that it was conditionable. It was found that forebrain ablation did not result in any loss in the ability of these operated fish to make a brightness discrimination. In fact, the operated animals learned the brightness discrimination more rapidly than normal animals. Furthermore, the forebrainless fish were able to generalize to another brightness problem. Control tests were run to substantiate this finding.

The blood-brain barrier may be demonstrated in mammals by the exclusion of certain dyes from the brain. In addition, anions such as thiocyanate, iodide, bromide, and sulfate are maintained in the brain at various brain/plasma ratios. In mammals, these ratios, in vivo have always been lower than the brain/plasma ratio for chloride. The present investigation was undertaken to study the blood-brain barrier of the goldfish by determining the entry of various dyes and ions into the brain parenchyma. The brains of the fish were examined 4 hr after intraperitoneal injection of 1% trypan blue, bromphenol blue, sodium phenolsulfonephthalein or sodium fluorescein. The dyes did not enter the brain parenchyma except at the anterior and posterior choroid plexus and the saccus vasculosus. The barrier could be disrupted by 70% alcohol or stab wounds in the brain. In addition, sodium thiocyanate was injected intraperitoneally or was introduced into the water of the aquarium. Tissue samples were taken 5–144 hr later and the thiocyanate space calculated. The chloride space of other fish was similarly calculated. The thiocyanate space of brain was significantly larger (p < 0.01) than the chloride space. The thiocyanate space of muscle was not significantly different from the chloride space. Since the thiocyanate space of brain exceeded the chloride space it is suggested that there are significant differences between the blood-brain barrier mechanisms of bony fish and mammals.

The effect of forebrain ablation on hue discrimination in fish has been studied. Cardiac deceleration, a conditioned autonomic response, was used as the measure of discrimination in a series of goldfish. Electric shock was the unconditioned stimulus. Forebrain-ablated fish were trained to a red and a green stimulus selected to be of equal brightness. These fish were subsequently tested on red and green stimuli of known brightness, as perceived by these experimental animals. Normal fish in this situation reacted to the test stimuli on the basis of their wavelength characteristics, but forebrainless animals reacted only to the brightness and not to the wavelength characteristics of the test stimuli. Other control tests substantiate this finding.

Two experiments were performed to determine whether regenerating preganglionic sympathetic fibers establish synapses randomly with any postganglionic neuron or selectively with specific postganglionic neurons. Experiment I . In normal cats the cervical sympathetic trunk (CST) is composed of nerve fibers deriving from the thoracic sympathetic rami T1 to T7. Of these rami, T1 contributes largely pupillodilator fibers and T4 contributes largely vasoconstrictor fibers (i.e., electrical stimulation of T1 produces pupillary dilation without decrease in ear temperature and stimulation of T4 produces a decrease in ear temperature without pupillary dilation). Following transection and regeneration of the CST, these relationships are re-established, indicating that the sympathetic preganglionics from T1 have established synapses preferentially with pupillary postganglionic cells and that the preganglionics from T4 have established synapses preferentially with vasomotor postganglionic cells. Experiment II . In normal cats pupillary dilation is elicited by electrical stimulation of rami T1 to T3, but not T4 to T7. However, stimulation of T4 to T7 does produce pupillary dilation 1 month after crushing T1 to T3 (i.e., before the crushed fibers have regenerated). This effect results from the formation of terminal collateral connections between the residual nonpupillary (T4 to T7) preganglionic nerve fibers and pupillary postganglionic cells (11). Six months postoperatively, pupillary dilation is elicited by stimulation of T1 to T3, but not T4 to T7. Apparently, after T1 to T3 fibers have regenerated, the nonpupillary collaterals (T4 to T7) become functionally inactive and the regenerated fibers again establish functional connections with the pupillary postganglionic cells. From these two experiments we conclude that there is a strong specific affinity between regenerating sympathetic preganglionic fibers and their appropriate postganglionic nerve cells.

Experiments were performed to determine if the fibers of the sciatic or tibial nerves that normally innervate the soleus and plantaris muscles will, after regeneration, selectively reinnervate these muscles. In addition to nine unoperated control rats, thirty-seven operated animals were studied 4 months after one of the following procedures: sciatic nerve transection and resuture in adults (9), sciatic nerve crush in adults (8), tibial nerve transection and resuture in adults (5), tibial nerve crush in adults (5), sciatic nerve transection in neonatal rats (5), sciatic nerve crush in neonatal rats (5). Recordings were made of the isometric tensions (T) developed by the soleus and plantaris muscles during supramaximal tetanic stimulation of lumbar nerves L4 and L5 singly and simultaneously (L4 + 5). In normal rats L4 supplies a proportionately greater functional innervation to the plantaris than to the soleus, the ratio TL4/TL5 being significantly higher in the plantaris than in the soleus. In none of the six operated groups could any such difference in the ratio of the two muscles be demonstrated. Consequently there is no evidence of selectivity in the regeneration of the plantaris and soleus nerve fiber constituents of the sciatic or tibial nerves. Both the plantaris and soleus muscles of each operated group showed greater tension overlap than the corresponding muscles of the unoperated control group. Tension overlap was computed as follows: (TL4 + TL5 − TL4 + 5) TL4 + 5 . Gold chloride impregnations of motor end plates indicate that the increased overlap results only in part from increased numbers of dually innervated (presumably bisegmentally innervated) muscle fibers.

Although the spinal cord of goldfish normally regenerates, 30 or more days of blockage by Teflon inserts will result in the loss of this regenerative capacity. The following studies were undertaken to investigate the ultrastructure of normal regeneration and the loss of regenerative capacity following Teflon blockage. The spinal cords of 13 normal and 27 cord-transected goldfish were examined with the electron microscope. Cord-transected goldfish were killed 1 day to 3 months postoperatively. In 15 additional goldfish the spinal cords were transected and Teflon sheet placed between the severed stumps for 1–3 months, after which time it is known the spinal cord will not regenerate following Teflon removal. The spinal cords were immersed in buffered 1% osmic acid and prepared for electron microscopy. In the absence of Teflon, regenerating axons were repelte with smooth endoplasmic reticulum and mitochondria. The majority of axons had regenerated across the site of lesion by day 30 and synapsed in the first segment of the caudal spinal cord stump. Dendrites regenerated in neurite fascicles along with axons. After blockage by Teflon, the nerve fiber tips grew into juxtaposition in zones subjacent to a glialependymal scar. These axons possessed multibulbous processes at the tip which usually formed axoaxonic and axodendritic synapses. Retransection (five goldfish) of the spinal cord one segment rostral to the Teflon block resulted in the degeneration of approximately 50% of all the synapses in the synaptic zone subjacent to the glial-ependymal scar.

Spinal cord hemisection results in limited regeneration of nerve fibers and the sprouting of intact nerve fibers proximal to the site of lesion. Electron microscopically the nerve fibers form new synaptic complexes. The following study was undertaken in the rat to determine the synaptic profile of neurons during the regenerative process. The spinal cords of 40 rats were hemisected at vertebral segment T-2 and 5 animals per group (in addition to 5 normals) were utilized 10, 20, 30, 45, 60, and 90 days posthemisection. Tissue was prepared by the Rasmussen technique for the light microscopic demonstration of boutons. In addition, five animals had the cord hemisected and the spinal cord tissue was prepared for electron microscopy 40 days postoperatively. Bouton counts were made on perikaryon and primary dendrite (of the same neuron) of neurons in lamina IV, lamina VII, and on motoneurons. Statistical analysis of counts from coded material was carried out utilizing a polynomial regression, analysis of variance, and a Neuman-Keuls a posteriori analysis. The number of boutons on the perikaryon of neurons on the operated side demonstrated a significant decrease in boutons at 10–20 days postoperative followed by a significant increase in boutons at 30 days to levels below normal innervation. This reinnervation was followed by a significant secondary loss of boutons from 30 to 60 days. This trend of bouton alteration was repeated (with minor variations) on the perikaryon of neurons on the unoperated side of the spinal cord. This general trend was repeated on the primary dendrite on both operated and unoperated side of the spinal cord. However, the number of boutons on primary dendrite usually returned to normal. The regenerated boutons (30 days) frequently contained granular vesicles (predominantly 900 A). The boutons that degenerated 30–60 days had all combinations of synaptic vesicles. This is an indication that former and new synaptic complexes were degenerating. The alteration of boutons on perikaryon and primary dendrite following hemisection suggests: that there is a critical period of initial denervation (0–20 days); that reinnervation occurs within the first 30 days; and that a secondary denervation occurs after 30–60 days. This recombination of neuronal circuits may represent an accelerated view of a dynamic continuum which imparts information to denervated neurons about altered neuronal circuitry adding a further dimension to the plasticity of the adult nervous system.

Single applications of puromycin into the lesion site of spinal hemisected rats were previously shown to induce transient nerve fiber growth into the lesion. We determined the protein incorporation of [ 3 H]lysine into rat spinal cord and brain treated with puromycin in groups of animals 3 h, 12 h, and 1, 3, 7, and 14 days after a left spinal cord hemisection at T2 and compared it to that in saline-treated controls. Each group consisted of two control animals, in which a left spinal hemisection was made at T2 and a Gelfoam sponge soaked in saline implanted in the lesion, and five experimental animals which were implanted with Gelfoam soaked in 1 m m puromycin in saline. One hour prior to utilization, animals were injected subcutaneously with 200 μCi l -[4,5( n )- 3 H]lysine monohydrochloride. In brain, no hemispheric or regional differences in protein or soluble fraction radioactivity could be detected in puromycin-treated or control animals. In spinal cord, inhibition of amino acid uptake was not demonstrable 3 h after hemisection, but could be detected at the site of the lesion 6 and 12 h after implantation. Three and 7 days after spinal hemisection, puromycin-treated animals exhibited a larger uptake of amino acid into protein at the lesion site than controls. The results suggest that morphological effects produced by puromycin on spinal cord regeneration may correspond to chemical events occurring within the first 24 h after cord hemisection and puromycin implantation.

Morphological aspects of the implantation of 11-day-old fetal (E11) rat cortex into regenerating peripheral nerves of adult rats were analyzed during the first 3 weeks after transplantation. On day 11 of gestation, donor fetuses were removed from one uterine horn of pregnant Sprague-Dawley rats and pieces of cortex (1.0 X 0.5 mm) were dissected from them for transplantation. Fetuses and normally delivered pups from the other uterine horn were used as age-matched controls. In 45 adult male Sprague-Dawley rat hosts (200 to 300 g), the epineurium of the nerve to the biceps femoris muscle was crushed and incised, the perineurium minced, and E11 donor cortex was injected into the nerve. At 1 to 7 days postimplantation (DPI), undifferentiated neuroepithelial cells were dispersed or in oval neuroepithelial-like structures throughout the regenerating nerve. At 7 to 14 DPI, neurons and neuroglia were differentiating and interspersed among fascicles of regenerating host axons. At 14 to 21 DPI, aggregates of differentiated neurons surrounded by neuropil containing a rich synaptic bed were interspersed with regenerated host peripheral axons. Fetal cortical implants survived, increased in size, and differentiated throughout the postoperative period, matching their timed controls.

Following hemisection of the rat spinal cord at the T1–T2 vertebral interface, dendritic sprouting was observed on neurons in the motoneuron pool at 45 days postoperative. The sprouting resulted in increased numbers of dendritic terminals, dendritic growth cones, and dendritic filopodial growth. The morphologic characteristics of the dendritic sprouts are similar to neurons in developing as well as in mature animals, and are correlated with synaptic renewal during spinal cord regeneration.

A single application of a protein synthesis inhibitor, puromycin, in the lesion site of a spinal hemisection transiently decreases protein incorporation for less than 24 h and results in axonal sprouting and dendritic and synaptic stability for at least 60 days. In this study we characterized the protein-altering properties of single applications of a different protein synthesis inhibitor, cycloheximide, for comparison. The protein incorporation of [3H] lysine into hemisected rat spinal cord (left side, T2) treated with cycloheximide (100 micrograms/ml in Gelfoam sponge placed in wound at time of lesion) was tested 3 h, 6 h, 12 h, 1, 3, 7, and 14 days later and compared with that in 0.9% saline in Gelfoam implants as normal controls (N = 54). One hour prior to utilization, the animals were injected subcutaneously with 200 mu Ci L-[4,5(n)-3H]lysine monohydrochloride. No treatment-related differences in brain radioactivity were noted at any time postoperation. In spinal cord, inhibition of amino acid uptake by cycloheximide was not demonstrable at 3 h after hemisection, but could be detected from 6 h to 1 day after implantation. During that time, the TCA-soluble radioactivity at the lesion site was greater than in control animals (P less than 0.05). These results suggest that as with puromycin, morphologic effects produced by cycloheximide on spinal cord regeneration may correspond to chemical events which occurred within the first 24 h after cord hemisection and cycloheximide treatment.

Poikilotherms that are stored in the cold often do not give normal visual responses. This experiment was undertaken to examine the electrophysiological and morphological changes in the retina during cold stress. Twenty-two goldfish (Carassius auratus) were placed in a refrigerated aquarium and the temperature reduced from 20 to 6 C (±2) for up to 31 days after which temperature was gradually returned to 20 C. During the period of cold stress and recovery, recordings were made of the electroretinogram, fast retinal potential, and tectal evoked response. Eyes of 22 animals were removed at various intervals for histological examinations. Electrical signals of nine animals were tested during cold stress and recovery to monitor for cold effects on retinal and visual pathway discharge. The temperature of two normal goldfish was reduced from room temperature to 5 C during 2 hr to control for the effect of temperature on visual signals. This resulted in minimal physiological changes. All visual signals nearly disappeared within 14 days of exposure to continuous cold stress. Slow recovery to 20–100% of normal was achieved within 52 days after return to room temperature. Statistical analysis of results from samples of retinal tissue indicated that electrical signal loss and return was paralleled by depletion and restitution of many cone outer segments. The return of visual behavior was correlated with the return of nearly normal complements of cone outer segments.

The regenerative capacity of the central nervous system appears to be related to phylogenetic level. The present studies in the goldfish and rat were undertaken to ascertain the regenerative capacity of the telencephalon. Five types of telencephalic lesions performed in goldfish were total removal, removal of one hemisphere, removal of lateral portion of hemispheres, partial section of hemisphere, and stab wound in hemisphere. In the rat a stab wound was made in occipital cortex. Animals were killed up to 2 years postoperatively. In the goldfish regardless of age at operation, removal of a mass of tissue did not result in restitution of the ablated parenchyma. After partial transection, cysts were observed in the telencephalic parenchyma partially or completely lined with pseudostratified columnar epithelial cells which were often ciliated. Giant pleomorphic reactive neuroglial cells were found in the area of the lesion. These cells usually became necrotic after 60 days. Severed axons regenerated around the lesions and reformed characteristic tracts. Ninety days after transection the area of the lesion was sometimes filled with epithelial cells which subsequently became glial in form so that the area of lesion was characterized by cysts and increased vascularity. Cortical regeneration in the rat was abortive. The cellular reaction to injury was compared in these two animals.

The role of the telencephalon in hue discrimination and interocular transfer of hue discrimination has been studied. Cardiac deceleration, a conditioned autonomic response, was used as the measure of discrimination in goldfish. Electric shock was the unconditioned stimulus. After occluding one eye, normal fish were trained to a red and a green stimulus selected to be of equal brightness. The naive or transfer eye was then tested on red and green stimuli of known brightness as perceived by these experimental animals. Normal fish and fish with the forebrain contralateral to the trained eye ablated demonstrated interocular transfer of a hue discrimination. Following ablation of the forebrain homolateral to the trained eye or bilateral forebrain ablation, operated fish demonstrated a loss in ability to make an interocular transfer to generalizations of hue. However, the ability to make an interocular transfer of a hue discrimination returns if a suitable period is allowed postoperatively. It was concluded that the telencephalon is not essential for color vision or for the interocular transfer of color vision in fish.

In the fish and amphibia the regenerative capacity of the spinal cord decreases from the embryonic stages of life to adulthood. The present experiments examine the relationship between age and the regeneration of severed spinal elements in the adult goldfish. The spinal cord of 1-, 2-, and 3-year old fish was transected in the mid dorsal fin region. The operation resulted in paralysis caudal to the lesion. Normal swimming and turning movements occurred in 20–25 days for 1-year olds, 25–30 days for 2-year olds, and 35–40 days for 3-year olds. In all age groups stimulation of the cervical spinal cord 30 days postoperatively elicited electromyographic responses in the myotomes caudal to the lesion. By 7 days post-operatively, small branching nerve fibers had grown from both proximal and distal stumps into the connective tissue scar in the gap. By 90 days postoperatively regeneration was complete. At this time there was a 26% decrease in cord diameter in the 2-year olds, a 28% decrease in the 3-year olds, but only 0.5% decrease in 1-year olds. In the region of transection the percentage of regenerated fibers was significantly greater in the 1-year olds than in the 2- and 3-year olds; in the 1-year olds the number of fibers was 90% of normal whereas in the 2- and 3-year olds it was 50% of normal. There were no motor horn cells in the regenerated area, and Mauthner's axon did not regenerate. It is concluded that spinal cord regeneration in the goldfish is age dependent; however, regeneration was sufficient in all age groups studied to result in complete restitution of function.