It is widely believed that damaged axons in the adult mammalian brain have little capacity to regrow, thereby impeding functional recovery after injury. Studies using fixed tissue have suggested that serotonin neurons might be a notable exception, but remain inconclusive. We have employed in vivo two-photon microscopy to produce time-lapse images of serotonin axons in the neocortex of the adult mouse. Serotonin axons undergo massive retrograde degeneration following amphetamine treatment and subsequent slow recovery of axonal density, which is dominated by new growth with little contribution from local sprouting. A stab injury that transects serotonin axons running in the neocortex is followed by local regression of cut serotonin axons and followed by regrowth from cut ends into and across the stab rift zone. Regrowing serotonin axons do not follow the pathways left by degenerated axons. The regrown axons release serotonin and their regrowth is correlated with recovery in behavioral tests.

The serotonin system in the CNS has been broadly implicated in the regulation of affect, aggression, sexual behavior, sleep, and cognitive function (). It originates in the raphe nuclei, a distributed series of cell groups with weakly defined boundaries flanking the midline of the midbrain and brainstem (). The rostral group of raphe nuclei, consisting of the supralemniscal nucleus, median raphe, and dorsal raphe, contains serotonin neurons that project widely to forebrain structures, including all regions of the neocortex, the striatum, olfactory bulbs, amygdala, and hippocampus. In the rodent, the axons of the rostral raphe nuclei are almost entirely thin and unmyelinated (). Those fibers that innervate the neocortex form a C shape when observed in the sagittal plane: they pass through the lateral hypothalamus and the basal forebrain, running in the medial forebrain bundle (MFB). The fibers then turn dorsally, elaborating branches that form projections to the frontal cortex, and then turn posteriorly to innervate parietal, temporal, and occipital cortices, respectively ( Figure 1 , upper right panel). Electron microscopy of neocortical serotonin axons shows that the vast majority of vesicle-bearing varicosities in serotonin axons have no corresponding postsynaptic density laden with serotonin receptors. This suggests that the action of serotonin on its targets is mainly produced by slow, diffuse volume transmission rather than fast, localized synaptic transmission ().

Axons that sustain damage in the adult mammalian brain are thought to fail to regrow, and this failure is believed, at least in part, to result from factors in the matrix of tissue that broadly inhibit axonal growth (). Rather, the limited functional rewiring that occurs after brain trauma is proposed to be dominated by the compensatory local sprouting of neighboring uninjured axons (). There have been suggestions from studies in both damaged brain () and damaged, grafted spinal cord () that axons of serotonin neurons might have an atypical capacity for regrowth. Following a lesion of serotonin axons in the rat neocortex with a high dose of amphetamine, there is a gradual reappearance of serotonin-immunoreactive axons over many months (). While suggestive, these fixed-tissue studies have been inconclusive. They were unable to distinguish degeneration and subsequent regrowth from a transient amphetamine-mediated downregulation of serotonin levels within intact axons. Nor have these studies been able to distinguish potential regrowth from damaged axons from compensatory sprouting of local undamaged axons. Thus, the regrowth of serotonin axons remains an important topic of debate that speaks to a central view in the recovery of neural function. Here, in order to observe the serotonin axon recovery process directly and disambiguate the results from fixed-tissue studies, we have performed in vivo two-photon time-lapse imaging over many months in adult serotonin transporter-EGFP BAC (bacterial artificial chromosome) transgenic mice () following several forms of injury.

Here, we have applied this technique to serotonin axons running in layer 1 of the neocortex and have found nearly identical results: in 7/7 mice, laser lesions of a single axon produced retraction over a distance of ∼10–30 μm followed by a complete failure to regrow from the severed end. Figure S11 shows an exemplar experiment in which a single serotonin-EGFP axon was ablated, leaving neighboring serotonin-EGFP axons intact (although presumably some neighboring unlabeled axons and other neuronal and glial structures are damaged as well). The surviving portion of the presumably proximal axon was swollen 1 day after the lesion, but at 4 days after the lesion had returned to a superficially normal appearance with typical diameter and varicosity density. However, the severed end of this axon was then entirely static, exhibiting no further elongation or retraction over a 16 week monitoring period. In sum, while profound serotonin axon regrowth was seen following PCA or neocortical stab lesions, ablation of a single serotonin-EGFP axon did not produce either regrowth from the severed end or sprouting of neighboring intact serotonin-EGFP axons. It is unclear why laser-axotomized serotonin axons fail to regrow while stab-lesioned axons succeed. One possibility is that the process of laser axotomy locally disrupts the structure of the serotonin axon or perhaps the extracellular matrix in a manner that persistently blocks regrowth. Another possibility is that serotonin axon regrowth requires a threshold level of damage to be evoked and the small laser lesions used herein are below this threshold.

Previous work has used a high-energy pulsed tuned infrared laser to sever individual GFP-labeled glutamatergic axons in the adult mouse neocortex (). Ablation of either pyramidal cell or thalamocortical axons with a laser microlesion produced a small degree of retraction of the proximal axon, followed by little or no regrowth from the severed end when monitored for up to an entire year. This failure to regrow was seen even though the microlesions did not create a persistent glial scar ().

A caveat should be sounded about the apparent cell-free rift in the tissue evident 1 hr after the stab lesion ( Figure 8 A). While similar rifts were observed in tissue sections from 4/4 mice, it is possible that these rifts are expanded by the mechanical stress of tissue slicing and processing. While it is likely that a cell-free zone is present at this point in vivo, its width may not be accurately measured in these fixed tissue sections.

When mice were sacrificed 1 week after the stab injury, the rift zone was still discernable as a region of diffuse NeuN immunoreactivity, but the tissue appears to have knit together to partially repair the rift. While there were 194 ± 9 axons in the rift volume in control mice (n = 8), and this was, of course, reduced to zero in mice sacrificed 1 hr after the stab injury (n = 4), progressive recovery was seen beginning at 1 week (25 ± 1 axons, n = 9 mice) and continuing at 3 months (62 ± 4 axons, n = 5) and 6 months (76 ± 3 axons, n = 10; Figure 8 B). An even greater degree of recovery was seen when using a measure that approximates total serotonin axon length within the rift (percent area occupied by axons in the rift zone; Figure 8 B). Growth of serotonin axons into the rift zone proceeded similarly in layer 1, which has very few neuronal cell bodies, and layer 5, which has many neuronal cell bodies.

To further test the hypothesis that serotonin axons regrow following neocortical stab injury, the same stab protocol was employed but was followed, at various durations, by EGFP immunohistochemistry to reveal serotonin axons together with NeuN immunohistochemistry, which labels neuronal nuclei. This technique relies on between-animal comparisons but has the advantage of allowing for visualization of all the cortical layers. Sagittal sections were prepared from a region of somatosensory cortex centered on the medio-lateral extent of the stab rift to avoid rift edge effects ( Figure 8 A). Sections from mice sacrificed 1 hr after the stab lesion reveal a broad rift ∼150 μm wide, devoid of tissue and extending from the brain surface through all six cortical layers to the underlying white matter. Within approximately 100 μm of the rift edge, on both sides, serotonin axons are mostly swollen and fragmented, while those farther away appear morphologically normal.

(B) The left panel shows a schematic representation of the serotonin neurons of the dorsal raphe and their C-shaped projection to the neocortex in the sagittal plane. The right panel shows the normalized area occupied by the serotonin-EGFP signal at various time points in stab injured and control mice within the rift zone in the somatosensory cortex (location 7).

(A) In serotonin transporter-EGFP mice, the serotonin axons were labeled with an antibody directed against GFP (green), and a subset of neurons was labeled with the nuclear marker NeuN (red) to provide landmarks. In stab-lesioned mice, serotonin axons were completely lost in the rift area at the 1 hr post-lesion time point and small number of axons started to enter the stab rift zone area 1 week after injury, becoming greater at later time points.

At 18 weeks after the lesion, 28/36 traced severed ends showed new growth ( Figures S9 C and S10 ). That growth ranged from 12 to 877 μm with a mean of 182 μm. Of the 28 severed ends of axons that showed new growth, 7 were able to traverse the stab rift completely and emerge on the other side. In 11/28 severed axons, the regrown portion of the axon exited the field of view, so the values here represent only a lower bound of the distance. There are also new segments of serotonin axon within the rift that cannot be traced to cut ends within the imaging volume. These may derive from sprouting of neighboring uninjured axons, or they may be growth from the ends of cut axons that are present just outside the imaging volume.

Is the extensive regrowth of serotonin axons seen following PCA lesion a general property of serotonin axons or some specific response to pharmacological injury that would not be seen with traumatic injury? To address this question, we employed a cortical stab protocol. Adult mice that had received craniotomies overlying the right somatosensory cortex were challenged by brief stereotaxic insertion to a depth of 2 mm of a miniature surgical blade, 1.2 mm wide. The blade was oriented with the width running in the coronal plane so that the resultant stab wound would transect most of the serotonin axons, which generally run in a rostrocaudal course in this region. Figure 7 A shows an exemplar mouse in which in vivo two-photon imaging of serotonin axons was performed immediately before and ∼1 hr after the stab injury. This treatment was followed by implantation of a cranial window and regular weekly monitoring for 18 weeks. One hour after stab injury, 36 randomly selected cut ends of axons were traced at the rift edge. By 1 week after injury, 25 of these cut ends displayed new axon growth, a value that increased to 29 and then 31 at 9 and 18 weeks, respectively. In addition, some of the regrown axons growing from the cut ends at the rift edge formed new branchpoints: there were 4, 20, and 27 new branchpoints at 1, 9, and 18 weeks after the stab lesion, respectively. When total axon length within the rift was measured for this mouse, it was 20.6 mm before the stab lesion and 0 mm ∼1 hr afterward. One week after the stab, there was 4.2 mm of axonal length present, and this value increased to 11.2 and 16.3 mm at 9 and 18 weeks, respectively ( Figure 7 B). In the population of six mice, in which serotonin axons were completely ablated within the stab rift, there was some recovery evident even after 1 week (17.8% ± 5.5% of baseline rift axon number) and substantial recovery after 18 weeks (81.0% ± 7.4%; Figure S9 ). Thus, the stab wound severs serotonin axons, but the cut ends of the axons do not appear to persistently regress more than a few microns. This stands in notable contrast to PCA treatment, in which the damaged axons regress beyond the field of view with in vivo imaging and likely regress all the way to the caudal medial forebrain bundle.

(A) Exemplar maximal z projection (top) and 3D axonal tracing images (bottom) after stab injury show significant degeneration of serotonin axons and subsequent recovery over 18 weeks. Purple and pink color axons in 3D images indicate degenerated and survived axons segments, respectively. Each of the many other colors codes for the week of first appearance of a new axon entering the field of view. Serotonin axons were highly stable in control mice.

Prior work has shown that attenuation of the serotonin system can result in behavioral hyper-reactivity (). We found that PCA treatment produced significant increases in the amplitude of acoustic startle responses in comparison to saline-treated controls, measured 1 week after treatment (saline, 85.3 ± 21.2 arbitrary startle force units in response to 120 db tone, n = 10; PCA, 143 ± 33.3 arbitrary startle force units in response to 120 db tone, n = 10; p = 0.00478; Figure 6 B). However, 6 months after treatment, no difference between the saline- and PCA-treated groups was found (saline, 70.3 ± 22.8 arbitrary startle force units in response to 120 db tone, n = 9; PCA, 58.8 ± 8.4 arbitrary startle force units in response to 120 db tone, n = 10; p = 0.872). Likewise, in a forced swim test, PCA treatment decreased the amount of time spent immobile in comparison to the saline group at 1 week after treatment (saline, 92 ± 6.8 s, n = 10; PCA, 64.7 ± 9.1 s, n = 10; p < 0.05; Figure 6 B). Again, 6 months after treatment, no difference could be seen between these groups (saline, 65.7 ± 9.7 s, n = 9; PCA, 62.4 ± 11.8 s, n = 10; p > 0.05). The degeneration of serotonin-EGFP axons by PCA treatment was confirmed with immunohistochemistry for both voltammetry and behavioral experiments (data not shown). Might these behavioral effects result from PCA damage to dopaminergic neurons? To determine whether PCA treatment also injured dopamine neurons, we performed immunohistochemistry using antibodies directed against tyrosine hydroxylase. No damage to dopamine neuron cell bodies in the ventral tegmental area or dopamine axons in the dorsal striatum or somatosensory cortex was observed ( Figure S8 ). Thus, 6 months after PCA treatment, when significant recovery of serotonin innervation has occurred ( Figures 2 and 3 ) and this recovery was sufficient to restore evoked serotonin release as determined using voltammetry ( Figure 6 A), both of these behavioral measures normalized to control levels ( Figure 6 B). Of course, the recovery of serotonin release and the recovery of these behavioral measures 6 months after PCA lesion are a mere correlation at this point, and there is no proof that they are causally related.

We have shown that the new axons that enter the field of view following PCA treatment are serotonin transporter immunopositive ( Figure S6 ), express a normal density of axonal varicosities, and recapitulate the overall spatial distribution of pre-lesion axons ( Figures 5 and S7 ). However, this does not prove that these axons have the capacity to release serotonin when action potentials invade. To address this question, a stimulating electrode was placed in the medial forebrain bundle of anesthetized serotonin-transporter-EGFP mice, and a carbon fiber microelectrode for serotonin fast-scan cyclic voltammetry (FSCV) () was placed in layer 1 of the somatosensory cortex. Serotonin release was evoked by a train of pulses (120 pulses at 60 Hz and 350 μA), as shown in the color plot in Figure 6 A. FSCV color plots represent a grouping of individual cyclic voltammograms, collected every 100 ms with current in false color. A cyclic voltammogram collected toward the end of the stimulation identifies serotonin via the discrete position of redox peaks ( Figure 6 A, inset). The current versus time taken at the peak oxidation current is directly convertible to serotonin concentration versus time. In saline-treated mice, peak evoked serotonin release was measured at 1 week (9.4 ± 1.5 nM, n = 2), 3 months (26.0 ± 6.4 nM, n = 2), and 6 months (14.5 ± 8.3 nM, n = 2) after treatment. Serotonin levels and release-uptake profiles are within the characteristic range previously reported in mice (). By contrast, no evoked serotonin release was detected 1 week or 3 months after PCA treatment (n = 6 and 4, respectively). However, 6 months after PCA treatment, a recovery of evoked serotonin release was evident (11.9 ± 2.9 nM, n = 4). By comparison, the total length of serotonin axons has recovered from its nadir of 25.5% ± 3.5% of baseline at week 1 to 53.8% ± 3.4% at 3 months and 68.0% ± 6.7% at 6 months. A likely reason for the lack of signal at 3 months is that evoked serotonin was below our limit of detection. Alternatively, new serotonin axons in the somatosensory cortex at 3 months after PCA are present but may require further maturation to release serotonin in response to electrical stimulation.

(B) Acoustic startle and forced swim test indicate hyper-reactivity 1 week after PCA treatment when compared with the saline-treated group (n = 10 for each group). At the 6-month time point, there was no significant difference between the saline (n = 9) and PCA (n = 10) groups for either test. ∗ p < 0.05.

(A) Representative color plot from saline-treated mouse (3 months) showing stimulation-evoked (120 pulses at 60 Hz) serotonin in the somatosensory cortex. Potential is on the abscissa, time is on the ordinate, and current is in false color. Stimulation of the MFB is indicated by the green bar under the color plot. Inset: a single cyclic voltammogram taken from the position of the vertical dashed line displaying characteristic serotonin redox peaks. Averaged, normalized [serotonin] evoked versus time traces are shown in the bottom panels. These are derived from the positions denoted by the horizontal dashed lines in the color plots shown in the middle panels. Stimulation is indicated by the green vertical bar.

Are there any morphological or positional criteria that can be used to predict which axons will survive PCA treatment? Axons at the week −1 pre-treatment time point were divided into two groups: those that would later survive PCA treatment and those that would degenerate. When these groups were compared, neither tortuosity nor varicosity density differed between them (tortuosity of surviving axons was 1.15 ± 0.01 compared with 1.16 ± 0.0 for those that would later degenerate; varicosity density of surviving axons was 0.44 ± 0.02 varicosities/μm compared with 0.42 ± 0.01 varicosities/μm for those that would later degenerate). In addition, the finding that 3D axon distribution density was not affected by PCA treatment (compare week −1 and week 1; Figure S7 ) suggests that an axon’s position in 3D space within our imaging volume was not a predictor of whether it would survive.

The location in 3D space of axons was calculated as a distribution of probability densities in three dimensions for both PCA- and saline-treated mice ( Figure S7 ). In saline-treated mice, these 3D axon distributions were remarkably stable across time (from −1 to 21 weeks), consistent with stable exemplar images ( Figure 3 ). In PCA-treated mice, these distributions were also remarkably similar, even though PCA produced a large reduction in axon density followed by a slow recovery. This consistency indicates that even though the new axons do not grow in the exact same locations where degenerated axons previously ran, overall, the new axons that underlie recovery after PCA recapitulate the 3D axon distribution density of the pre-lesion state.

When new axons enter the field of view, do they grow along pathways that had previously been established by degenerated fibers? Casual observation of the trajectories of new axons suggests that they do not ( Figure 3 A). To quantitate this impression, an overlap index was calculated in which each new traced axon at week 27 was separately scored for overlap in 3D space with each traced degenerated axon present at week −1 in two different PCA-treated mice. We set a criterion that >80% of the points along a new axon must overlap those of a degenerated axon in order for the axon to be scored as overlapping. We further set the overlap scoring zone to have a radius of 4.5 μm around each point in the new axon to account for small changes in the geometry and orientation of the imaged volume over 29 weeks. This analysis showed that only 3% of the new axons overlapped with degenerated axon pathways. These parameters are valid because when they are applied to comparing surviving axons at week 27 with the same axons at week −1, 96% of the surviving axons are scored as overlapping. Thus, very few axons grow along pathways abandoned by degenerating axons. Indeed, it is possible that even the 4% positive score for new axons represents new axons are that are growing close to, but not precisely along, the pathways of degenerated axons, since the limits of our imaging technique do not allow us to make this distinction. Importantly, these results further argue against a model in which intact serotonin axons are merely depleted of serotonin by PCA treatment (or depleted of EGFP in Slc6a4-EGFP mice) and then slowly refill, as this mechanism would be expected to yield a high degree of overlap between new axons and “degenerated” axon pathways.

When new axons enter the field of view after PCA treatment, they generally recapitulate the morphology of both surviving axons and control saline-treated axons. Figure 5 C shows that the tortuosity of new axons is similar to that of these other populations (tortuosity ratio of new axons at week 19 is 1.18 ± 0.02 compared with 1.16 ± 0.04 for surviving PCA-treated axons at week 19 and 1.12 ± 0.01 for surviving saline-treated axons at week 19). Likewise, the density of axonal varicosities in new axons is similar to surviving axons and saline-treated controls (data for week 19 are as follows: survived, 0.43 ± 0.01 varicosities/μm; new, 0.42 ± 0.01 varicosities/μm; p = 0.58).

Examination of exemplar images indicates that the new axons repopulating the field of view following PCA treatment do not grow along surviving axons, nor are they guided by blood vessels ( Figures 3 and 4 ). The former conclusion is supported by an analysis of axon neighbor relations ( Figures 5 A and 5B ). When the distance to the nearest axon is calculated for every point along every axon in the field of view, we found that PCA treatment increases the mean distance to the nearest neighbor by a factor of 2.39 ± 0.18 at week 1 (compared to the pre-treatment time point at −1 week), and this distance decreases gradually as the field of view is repopulated with new axons (a factor of 1.36 ± 0.07 at week 19). Importantly, a histogram showing the distribution of nearest neighbor distances fails to show a peak at distances <1 μm at any time point, indicating that serotonin axons rarely contact each other, as would be expected if the surviving axons served as a scaffold for new axons. In fact, the changes in the nearest neighbor histogram after PCA treatment are most consistent with a model in which new axons actively avoid contact with surviving axons, sprouting axons, or each other.

(B) Histograms of the distance to nearest neighbor for selected weeks. At week 1, the lower axonal density is reflected by an increase in each axon’s distance to its nearest neighbor. By week 27, as axons regrow, the distance to nearest neighbor is similar to control (week −1).

(A) Average distance to nearest neighbor for all points in the axonal tracing, normalized to the average distance to nearest neighbor for week −1. In PCA-treated mice (left panel), the distance to nearest neighbor approaches control levels as axons regrow. In saline-treated mice (right panel), the distance to nearest neighbor remains stable. The shaded area represents the SE.

Might the stress of PCA treatment cause Slc6a4-EGFP neurons to change their transmitter phenotype? To address this question, we performed in vivo time-lapse imaging weekly following PCA treatment, sacrificed the mouse, and processed the somatosensory cortex for immunohistochemistry with an antibody directed against the serotonin transporter. When the field of view from the in vivo imaging was recovered, it was revealed that surviving, sprouted, and new axons were all serotonin transporter immunopositive ( Figure S6 ), arguing against a change in neurotransmitter phenotype.

The new axons that repopulate the field of view are not transient structures: of the new axons present 6 weeks after PCA treatment, 100% are still present at week 19. Thus, it appears that recovery of serotonin axons does not result from overgrowth followed by pruning, at least within our 29-week-long maximum monitoring period. This pattern is similar to the initial development of serotonin fiber innervation of the neocortex in late embryonic and early postnatal life, which also lacks significant overgrowth and pruning ().

Are the new axons that appear in the imaged volume after PCA treatment growing from the shafts or severed ends of damaged axons, or are they axons that have sprouted from survivors adjacent to our field of view and have then grown to invade the imaged volume? While we cannot make definitive statements about events outside of the field of view, numerical considerations make this latter scenario unlikely. Within the imaged volume, 13 weeks after PCA treatment, there is an average of 123.6 ± 14.2 new axons, but only 15.4 ± 2.7 sprouted axons, and of those sprouted axons, only 4.4 ± 0.9 exit the imaged volume. Because some sprouted axons branch and both branches can exit the imaged volume, the average total number of sprouted axon exit events is slightly higher, 5.3 ± 1.2. Thus, for sprouted axons outside of the imaged volume to account for the new axons we see invading the imaged volume, the sprouting rate in adjacent regions would have to be consistently ∼23-fold higher than in the imaged volume. While this is formally possible, we favor the more parsimonious explanation, consistent with the immunohistochemical images of the entire forebrain ( Figures 1 and 2 ), that new axons originate from deeper structures and represent some form of long-distance regrowth. What we can’t determine definitively is whether these new axons ultimately originate from the neurons that sustain damage or from uninjured neurons.

Most of the new serotonin axons appearing after PCA treatment completely traverse the imaged volume from one weekly time point to the next. This is consistent with the overall rostrocaudal course of serotonin axons in neocortical layer 1 (). However, in some cases, new axons can be observed to gradually extend within the field of view from week to week, as shown in Figure 4 . Sprouting axons can also be seen to gradually extend within the field of view.

The top panels show an exemplar new axon. The new green axon appeared at week 12, and it continued extending through week 18. The bottom panel shows an exemplar sprouting axon. The pink axon in week 1 has survived PCA treatment. The sprout that extends from this surviving axon is first seen in week 6, and it gradually extends through week 9. For both the new and sprouting exemplars, the rightmost panels show a tracing of all axons and the maximal z projection to provide context. Population data are presented as mean ± SEM.

To assess the dynamics of axonal regrowth and allow for within-animal comparisons, we employed chronic in vivo two-photon microscopy. Here, we define regrowth as growth of axons in response to injury, irrespective of the cellular origin of growth (that is, either from damaged or undamaged axons). Cranial windows were implanted overlying the somatosensory cortex of serotonin transporter-EGFP mice. After an 11-day recovery period, a z stack encompassing layer 1 was acquired, followed by a second pre-treatment image taken 3 days later. Then, mice were injected with either PCA (n = 9) or saline (n = 4). Imaging of the same region was then continued at weekly intervals for as long as possible, ranging from 13 to 29 weeks. Figure 3 A shows exemplar maximal z stack projections and 3D axon tracings for PCA- and saline-treated mice. When total axonal length was calculated for each time point, it was revealed that PCA treatment produced a large decrease measured 1 week after treatment (25.5% ± 3.5% of baseline, mean ± SEM, n = 9), while the saline group was unaffected (98.0% ± 1.6%, n = 4). The PCA-treated group showed a slow, gradual recovery (64% ± 6.2% at t = 19 weeks, n = 4) that can be appreciated in a movie showing 3D projections and rotations of exemplar analyzed volumes ( Movie S1 ). In contrast, total axonal length of the saline-treated group was highly stable (91.8% ± 4.8%, t = 19 weeks, n = 3). Similar results were found when the total number of axonal branch points was scored ( Figure S5 ). To further analyze the recovery process, axonal segments were assigned to several groups: surviving axons, which persisted after treatment; sprouted axons, which grew as new branches elaborated from those axons that survived; and new axons, which only appeared in the field of view at some point after treatment. Figure 3 B shows that the slow recovery of total axonal length following PCA treatment is almost entirely due to the presence of new axons. The rate of local sprouting from surviving axons following PCA is very low (5.2% ± 1.1% at t = 19 weeks, n = 4) and is comparable to the rate seen in saline-treated control mice (4.3% ± 1.6% at t = 19 weeks, n = 3; Figure 3 B). The axons that survive PCA treatment are not merely dying slowly: of the surviving axons present 1 week after PCA, 96.5% are still present at week 19, slightly more than the 81.7% survival rate of saline-treated axons over this same period.

(A) Exemplar maximal z projection (top) and 3D axonal tracing images (bottom) after PCA treatment show massive degeneration of serotonin axons and subsequent slow recovery over 29 weeks. Purple and pink color axons in 3D images indicate degenerated and survived axons, respectively. Each of the many other colors codes for the week of first appearance of a new axon entering the field of view (for example, new axonal segments appearing in week 6 are orange and those appearing in week 11 are red). Serotonin axons were highly stable in response to saline treatment. The tubular structures composed of many horizontally elongated puncta in the z stack images are blood vessels filled with serotonin transporter positive platelets (). These blood vessels are indicated with yellow arrows.

As a further test of the possibility that apparent serotonin axon degeneration was merely an artifact of reduced Slc6a4-EGFP expression in intact axons, we injected the virus AAV5-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-pA into the dorsal raphe of serotonin transporter-Cre mice to produce surface membrane labeling () of the complete extent of dorsal raphe serotonin neurons, including their axonal projections to the neocortex. Following a 1 month period to allow for expression and transport of EYFP, mice received PCA or saline treatment and were then sacrificed 1 week or 3 months thereafter. This was followed by triple-label immunohistofluorescence to reveal EYFP and serotonin transporter expression in axons of the frontal and somatosensory cortex, as well as NeuN to reveal neuronal nuclei ( Figure S4 ). Because Cre recombinase activity is irreversible, this treatment yields a mouse in which EYFP expression is no longer subject to regulation, being under the control of the strong, ubiquitous EF1a promoter rather than the promoter of the serotonin transporter gene. In layer 1 of somatosensory cortex, in a saline-treated mouse sacrificed 1 week later, 23.7% ± 1.0% of the area was occupied by serotonin transporter immunoreactivity and 12.3% ± 0.9% of the area was occupied by EYFP immunoreactivity (n = 8 mice). Of the EYFP-positive pixels, 79.6% ± 3.9% were also serotonin transporter positive. This indicates that virally introduced EYFP selectively labeled approximately half of the serotonin transporter-positive axons. Importantly, 1 week after PCA treatment, serotonin transporter immmunoreactivity was reduced to 7.0% ± 0.9% and EYFP immunoreactivity was reduced to 3.6% ± 0.3% (n = 6). When these measurements were repeated on a cohort of mice allowed to recover for 3 months after PCA treatment, we observed partial recovery in both measures (serotonin transporter, 14.2% ± 1.0%; EYFP, 8.1% ± 0.4%, n = 9), similar to the degree of recovery seen using Slc6a4-EGFP mice ( Figure 2 ). These results argue that PCA treatment does indeed cause massive degeneration of serotonin axons rather than merely appearing to do so through an effect on EGFP expression in Slc6a4-EGFP mice.

These findings are consistent with previous reports using amphetamine treatment in rats followed by immunohistochemistry with antibodies directed against serotonin (). However, because here the serotonin axons are visualized with an antibody to untethered cytoplasmic EGFP, the present results argue against a model in which intact serotonin axons are merely depleted of serotonin by PCA treatment and then slowly refill. Could PCA treatment somehow lead to a reduction of EGFP expression driven by the promoter of the serotonin transporter? As one way to address this question, we measured serotonin transporter immunoreactivity in proximal, undegenerated axons coursing through the linear nucleus of the raphe complex, 1 week after saline or PCA treatment. No difference in serotonin transporter immunoreactivity was seen between saline and PCA treatment in these images (saline, 55.8% ± 1.7% area occupied by serotonin transporter signal; PCA, 60.1% ± 6.7% area, n = 3/group). By contrast, PCA treatment produced a profound decrease in serotonin transporter immunoreactivity in the same distal locations where EGFP signal was lost, such as somatosensory cortex (saline, 32.9% ± 1.4% area occupied by serotonin transporter signal; PCA, 4.3% ± 0.4% area, n = 7/group).

To visualize serotonin neurons, we have used a BAC transgenic mouse in which the promoter of the serotonin transporter drives expression of soluble EGFP to produce a cytoplasm-filling marker almost completely restricted to serotonin neurons in the adult brain (Slc6a4-EGFP mouse;) as indicated by EGFP + serotonin double-label immunohistochemistry ( Figure S1 , available online). Initially, we have sought to characterize the effects of amphetamine treatment using immunohistochemical techniques. Mice received eight intraperitoneal injections over 4 days of either para-chloroamphetamine (PCA; 20 mg/kg), a particularly toxic amphetamine derivative (), or saline. After a recovery period ranging from 1 day to 6 months, the mice were sacrificed and sagittal slices near the midline were processed for immunohistochemistry using antibodies against EGFP, to show serotonin neurons, and NeuN, to reveal a subset of neuronal cell bodies. Figure 1 shows exemplar confocal images taken 1 day after PCA treatment, revealing swollen serotonin axons in neocortical regions and the rostral MFB (see also Figure S2 for in vivo imaging of swollen axons 1 day after PCA treatment). There were no swollen axons seen in the more proximal portions of the axonal trajectory, including those that pass through the linear nucleus of the raphe, the posterior hypothalamus, and the more caudal portions of the MFB (data not shown). This pattern is consistent with the early stages of retrograde axonal degeneration and is congruent with well-established literature on amphetamine-induced degeneration of serotonin axons in rats, which has gone to great lengths to demonstrate the hallmarks of degeneration, including swollen, irregular axons (), and has used immuno-electron microscopy to show amphetamine-evoked destruction of microtubules and degenerated mitochondria in such axons (). Figure 2 shows representative images taken 1 week after PCA or saline treatment, starting with the serotonergic cell bodies of origin in the dorsal raphe nucleus, continuing with their long C-shaped projection through the MFB, and ending in the occipital cortex. These images reveal that PCA treatment produced a significant loss of serotonin axons in all of the cortical regions examined as well as in the rostral aspect of the MFB. Importantly, neither the cell bodies of origin in dorsal raphe ( Figures 2 A and 2B) nor the initial portion of the axons running in the linear nucleus of the raphe or the posterior hypothalamus was affected by PCA treatment, while the caudal portion of the MFB showed a small degree of axonal loss. When the period after PCA treatment was extended to 3 and 6 months, substantial recovery of EGFP immunoreactivity was observed in the MFB and neocortex ( Figures 2 C and S3 ).

(B) Serotonin cell body density in the dorsal raphe was not affected by PCA treatment in either short- or long-term measurements. Please note that the indicated times are after the end of the 4-day-long PCA treatment. Population data are presented as mean ± SEM.

(A) In serotonin transporter-EGFP mice, the serotonin axons were labeled with an antibody directed against GFP (green), and a subset of neurons were labeled with the nuclear marker NeuN (red) to provide landmarks. The central panel shows a schematic representation of the serotonin neurons of the dorsal raphe and their C-shaped projection to the neocortex in the sagittal plane. Each location along the pathway shows images taken 1 week after PCA or saline treatment. In PCA-treated mice, serotonin axons were lost in the most distal part of the projection, including the occipital (8), somatosensory (7), and frontal (6) cortex, as well as the rostral median forebrain bundle (MFB, 5). Serotonin cell bodies in dorsal raphe (1), axons in linear nucleus of raphe (2), and posterior hypothalamus (3) were unaffected.

One possible molecular explanation (of many) is that unlike the growth cones of glutamate axons, serotonin axonal growth cones fail to receive the stop signals from the tissue matrix that inhibit other axonal types. Ultimately, single-cell expression profiling of pre-lesion and regrowing serotonin neurons in the raphe complex will be useful to address this question, as will inducible deletion in the adult brain of candidate axon growth-promoting genes within serotonin neurons. Perhaps, uncovering the unique molecular properties of regrowth-competent serotonin axons and the discovery of genetic and pharmacological manipulations that enhance or suppress long-distance serotonin axon regrowth will allow for the development of therapies to promote axon regrowth in a wide variety of cell types.

Why are serotonin axons able to regrow in the adult brain when almost all other axons fail? One functional explanation is that since serotonin axons in the brain mostly operate through slow, diffuse volume transmission rather than fast, localized synaptic transmission, constraints on the spatial fidelity of regrowth are relaxed: unlike a glutamate-releasing axon, which forms conventional synapses, a serotonin-releasing axon needs only to achieve reinnervation of its approximate former position in order to restore functional volume signaling.

The inflammatory response produced by amphetamine lesion is transient and, unlike concussive or penetrating brain injury, no glial scar is produced (). Nonetheless, it appears that serotonin axons have the ability to penetrate glial scars in the brain (such as those produced following a thermal lesion;) and hence are likely to demonstrate regrowth and recovery of function following a broad array of brain injuries. Our own findings show that stab injury produces local astrocyte activation, as indexed by GFAP immunoreactivity, that persists for at least 10 weeks, and that regrowing serotonin axons crossing the stab rift can run adjacent to these activated astrocytes ( Figure S12 ). This finding is consistent with the previous observation in raphe spinal serotonergic axons that occasional regenerating axons can pass through GFAP-positive tissue bridges that form following complete spinal transection (). One important implication of these findings is that the tissue matrix of the adult CNS, generally thought to be non-permissive for axonal growth (), fails to inhibit regrowth of serotonin axons.

The regrowth of serotonin axons following a neocortical stab extends these findings in two important ways. First, it establishes that regrowth of damaged serotonin axons is not a unique feature of amphetamine lesions. Second, because the damaged axons almost entirely remain within the field of view for in vivo imaging, this allows us to assess the state of the severed ends of damaged axons. Here, the result is clear. A large fraction (>80%) of the severed ends of serotonin axons display new growth, and in many cases this regrowth is sufficient to completely cross the stab rift. Thus, the long-held view that axons in the brain fail to undergo regrowth following injury does not hold for serotonin neurons that innervate the neocortex.

Surprisingly, sprouting of uninjured axons within the field of view does not substantially contribute to the recovery of serotonin axon density following PCA lesion. In fact, the rate and extent of local sprouting is the same in PCA-treated and control serotonin axons. The particular serotonin axons that survive PCA treatment cannot be predicted on the basis of their location, neighbor relations, tortuosity, or varicosity density.

New axons that emerge following PCA lesion do not demonstrate overgrowth or pruning. They persist for at least 6 months. Furthermore, at the 6 month post-lesion time point, they have the capacity to release serotonin in response to electrical stimulation. These findings are consistent with previous reports showing that axons of serotonin neurons in the caudal raphe complex are among the first to cross a grafted and chondroitinase-treated spinal cord lesion in mouse models and can contribute to restoration of locomotion (), bladder, and diaphragm control ().

The present work shows that serotonin axons in the brain have the unusual property to undergo regrowth following damage produced by either a systemic amphetamine lesion or a local stab lesion. Following PCA lesion, the new axons largely recapitulate the morphology (varicosity density, tortuosity), neighbor relations, and layer-specific distribution of pre-lesion axons. Like uninjured axons, they are serotonin transporter immunopositive. However, unlike regrowing axons in the peripheral nervous system, they do not follow pathways in the tissue matrix that are left behind by previously degenerated axons.

Experimental Procedures

Transgenic Mice Slc6a4-EGFP BAC transgenic mice were made by Charles Gerfen (NIH) as part of the GENSAT consortium (line RP23-39F11, BAC BX86, RRID: MMRRC_030692-UCD). Slc6a4-EGFP BAC-Cre mice created by the GENSAT consortium and purchased from the Mutant Mouse Regional Resource Center at UC Davis (stock #031028-UCD and #017260-UCD, RRID: MMRRC_031028-UCD and MMRRC_017260-UCD). All experiments were approved by the Animal Care and Use Committees of Johns Hopkins University, or for the voltammetry studies, Wayne State University.

Surgical Procedure for In Vivo Imaging A craniotomy was made overlying the right somatosensory cortex in adult Slc6a4-EGFP mice, and serotonin axons in layer 1 were imaged with a two-photon microscope using 920 nm laser illumination. Mice aged 4–5 months were anesthetized with 1%–2% isoflurane and placed in a stereotaxic device (Stoelting). A small dose of dexamethasone (0.04 mL at 2 mg/mL) was administered by subcutaneous injection prior to surgery to minimize potential swelling at the surgical site, and a subcutaneous injection of buprenorphine (0.3 mg/mL) was also given before surgery to alleviate pain. The skull was exposed with a midline scalp incision, and a region (2 × 2 mm) over somatosensory cortex based on stereotaxic coordinates was carefully removed using a #11 surgical blade (the medial, anterior corner of the craniotomy was located 0.5 mm posterior and 1.0 mm lateral to bregma). During cutting of the craniotomy, the skull was bathed in saline to minimize damage to underlying structures. Surgifoam (Johnson & Johnson) soaked in ACSF was used to stop bleeding from the surrounding tissue. A square fragment of coverslip glass was placed inside the walls of the craniotomy, and Metabond cement (Parkell) was applied at the edges to form a cranial window and to fasten a laterally protruding stainless steel plate. Following surgery, Baytril (2.5 mg/kg, delivered by subcutaneous injection) was given as antibacterial prophylaxis.

In Vivo Imaging of Serotonin Axons For in vivo imaging, mice were anesthetized with 1%–2% isoflurane and placed on a feedback-controlled warming pad. The stereotaxic microscope stage was equipped with lateral bars that fastened to the mouse’s headplate for stabilization. The stage was fixed on an x-y translator under a laser-scanning confocal microscope (Zeiss LSM 510 NLO) equipped with a 40× W Plan-Apochromat VIS-IR water immersion objective (Zeiss, 1.0 NA) and a non-descanned photomultiplier tube attached to the epifluorescence port. Two-photon excitation (920 nm) was provided by a Chameleon vision II mode-locked Ti-Sapphire laser (Coherent). z stacks were acquired starting at the cortical surface and continuing to a depth of ∼100–200 μm with a step size of 1 μm. Each stack was imaged with a resolution of 1,024 × 1,024 pixels (0.31 μm/pixel; pixel dwell time = 3.2 μs).

PCA and Saline Injection Protocols Male and female mice aged 4–5 months were given PCA (Sigma-Aldrich; 20 mg/kg) or saline intraperitoneally in single-housed standard cages maintained at an ambient temperature 25°C–26°C with continuous airflow. Injections were performed two times per day for 4 days. On a single day, the injections were given 6 hr apart, and the interval between the second injection on one day and the first injection on the next day was 16 hr.

Virus Injection Serotonin axons were labeled by dorsal raphe injection of AAV5-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-pA. Adult serotonin transporter cre mice (4–5 months old) were anesthetized with 1%–2% isoflurane and placed in a stereotaxic device (Stoelting). A glass pipette with 40 μm tip diameter was filled with mineral oil and connected to Nanoject II (Drummond Scientific Company). The tip of the glass pipette was then front-filled with ∼1 μL AAV5-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-pA. Injections were made 4.7 mm posterior from bregma, at a depth of 2.8–3.2 mm. A dye volume of 0.1–0.2 μL was delivered with a Nanoject II over a 10 min period. The pipettes were left for 15 min before they were withdrawn. The virus was developed in Karl Deisseroth’s lab ( http://www.med.unc.edu/genetherapy/vectorcore/research-grade/in-stock-aav-vectors/deisseroth ) and purchased from UNC Vector Core.

Stab Injury A craniotomy was made overlying the right somatosensory cortex in adult Slc6a4-EGFP mice as described. A miniature blade (Surgistar, USM 6700) was placed on the dura of the somatosensory cortex with a stereotaxic device in a location that was roughly centered in the window but chosen to avoid major surface blood vessels. The blade was then slowly advanced into the brain to the depth of 2 mm. Surgifoam (Johnson & Johnson) soaked in ACSF was used to stop bleeding from the surrounding tissue. A square fragment of coverslip glass was placed inside and sealed with Metabond dental cement (Parkell).

Laser Axotomy Laser-induced axon ablation was achieved by focusing a parked pulsed two-photon laser beam on a spot at a depth of 50–100 μm beneath the pial surface of the somatosensory cortex and illuminating for 1 s. The wavelength used for laser axotomy was 850 nm and the power at the back aperture of the objective was ∼100 mW. Lesion locations were chosen to avoid blood vessels, and a single lesion was made per mouse.

Immunohistochemistry The mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and perfused intracardially with PBS followed by 4% paraformaldehyde in PBS at 4°C. The entire brain was removed and fixed in 4% paraformaldehyde for 3 hr at room temperature and then cryoprotected in 15% sucrose in PBS overnight at 4°C, followed by a switch to 30% sucrose on the next day and continuing overnight. Sections of the mouse brain (40 μm thick) were prepared using a microtome and were washed with PBS and then blocked with 5% normal goat serum (Jackson ImmmunoResearch #005-000-001, RRID: AB_2336983 ) and 0.3% Triton X-100 in PBS for 2 hr at room temperature. The sections were incubated overnight at 4°C in primary antibody diluted in blocking buffer. The primary antibodies used were rabbit anti-5HT (1:20,000, ImmunoStar #20080, RRID: AB_572263 ), guinea pig anti-SERT (1:1,000, Frontier Institute #HTT-GP-Af1400, RRID: AB_2571777 ), rabbit anti-TPH2 (1:800, Millipore #ABN60, RRID: AB_11212793 ), rabbit anti-TH (1:1,000, Millipore #AB152, RRID: AB_390204 ), mouse anti-NeuN (1:500, Millipore #MAB377, RRID: AB_2298767 ), mouse anti-GFP (1:5,000, Life Technologies #A11120, RRID: AB_221568 ), chicken anti-GFP (1:5,000, Aves Labs #GFP-1010, RRID: AB_2307313 ), and rabbit anti-glial fibrillary acidic protein (1:1,000, Millipore #AB5804, RRID: AB_10062746 ). The sections were then washed with PBS and incubated in the secondary antibody in blocking buffer for 2 hr at room temperature. The secondary antibodies used were Cy3-labeled goat anti-rabbit (1:800, Jackson ImmunoResearch #111-165-003, RRID: AB_2338000 ), Cy3-labeled goat anti-guinea pig (1:800, Jackson ImmunoResearch #106-165-003, RRID: AB_2337423 ), Cy3-labeled goat anti-mouse (1:800, Jackson ImmunoResearch #115-165-062, RRID: AB_2338685 ), Cy5-labeled goat anti-mouse (1:200, Jackson ImmunoResearch #115-175-146, RRID: AB_2338713 ), FITC-labeled goat anti-mouse (1:800, Jackson ImmunoResearch #115-095-003, RRID: AB_2338589 ), and Alexa Fluor 488-labeled goat anti-mouse (1:1,000, Life Technologies #A11039, RRID: AB_142924 ). Then we mounted the sections on slides, and images were acquired using a single-photon confocal microscope (Zeiss).

Fast-Scan Cyclic Voltammetry Mice were terminally anesthetized with urethane (25% dissolved in 0.9% sodium chloride). Mouse body temperature (37°C) was maintained by placing a heating pad (Braintree Scientific) under the mouse throughout the duration of the experiment. A bipolar stimulating electrode (Plastics One) was implanted into the MFB (anteroposterior [AP], −1.58; medial lateral [ML], 1.10; dorsal ventral [DV], −4.8 – 5.0), and a Nafion-coated carbon fiber microelectrode was lowered into the somatosensory cortex (AP, −1.0; ML, +2.0; DV, −0.2 – 0.5). An Ag/Ag Cl reference electrode was placed into the contralateral hemisphere. The carbon fiber microelectrode was prepared thus: a single carbon fiber (T- 650; diameter, 7 μm; Goodfellow) was aspirated into a glass capillary (external diameter, 0.6 mm; internal diameter, 0.4 mm; A-M Systems), which was then pulled apart under heat and gravity. The protruding portion of the carbon fiber was cut under an optical microscope to a length of 50–100 μm. An electrical connection was forged with conductive epoxy, and Nafion electroplating was applied. A PCIe-6341 DAC/ADC card (National Instruments) generated an electrochemical waveform (1,000 V s−1 at 10 Hz from −0.1 V to 1.0 V, resting at 0. 2 V). A total of 120 biphasic electrical pulses were delivered to the stimulating electrode through a linear constant current stimulus isolator (NL800A Neurolog, Digitimer Ltd) at 60 Hz, 350 μA, and 2 ms per phase. A CHEM-CLAMP potentiostat (Dagan Corporation) measured output current. Data were collected and analyzed using WCCV 3.0 (Knowmad Technologies LLC). Data were background subtracted to remove a large capacitative current and then filtered at zero phase using a fourth-order Butterworth with a low pass of 5 kHz. A standard calibration factor for the serotonin waveform was utilized to convert current to serotonin concentration.

Acoustic Startle Response Two identical startle chambers (San Diego Instruments Inc.) were used for measuring startle reactivity and plasticity. Each mouse was placed in a Plexiglas cylinder (2 cm in diameter) within each chamber. A loudspeaker mounted 24 cm above the cylinder provided broadband background noise and acoustic stimuli. Presentations of the acoustic stimuli were controlled by the SR-LAB software and interface system (SR-LAB 94.1.7.48), which also rectified, digitized, and recorded responses from the accelerometer. The maximum voltages within 40-ms reading windows, starting at stimulus onset, were used as the measures of startle amplitudes. Sound levels were measured inside the startle cabinets using a digital sound level meter (Realistic, Tandy). The accelerometer sensitivities within each startle chamber were calibrated regularly and were found to remain constant over the test period. Each acoustic startle experiment consisted of several sessions. The startle responsiveness session was used to evaluate the effects of treatment on startle responsiveness and included a 5 min acclimatization period to a 70-dB background noise (continuous throughout the session), followed by the presentation of five 40-ms-long 100-, 110-, or 120-dB white noise stimuli presented in a randomized manner with randomized intervals ranging from 5 to 25 s. The data of the startle responsiveness session were analyzed using two-way repeated-measures ANOVA with treatment and amplitude of tone as independent variables and the amplitude of the startle as the dependent variable.

Forced Swim Test Mice were placed into glass cylinders, 15 × 30 cm, filled with room temperature water to a level of 20 cm. Mouse behaviors were video recorded for 6 min. The records were analyzed by a blind observer for the duration of immobility defined as the absence of movements or the paddling with one hindleg to keep balance on the surface. The time of immobility was assessed for the last 4 min of the test using StopWatch+ software (CBN, Emory University).