Tlx3 Exerts Direct Control in Specifying Excitatory Over Inhibitory Neurons in the Dorsal Spinal Cord.

The spinal cord dorsal horn is a major station for integration and relay of somatosensory information and comprises both excitatory and inhibitory neuronal populations. The homeobox gene Tlx3 acts as a selector gene to control the development of late-born excitatory (dILB) neurons by specifying glutamatergic transmitter fate in dorsal spinal cord. However, since Tlx3 direct transcriptional targets remain largely unknown, it remains to be uncovered how Tlx3 functions to promote excitatory cell fate. Here we combined a genomics approach based on chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) and expression profiling, with validation experiments in Tlx3 null embryos, to characterize the transcriptional program of Tlx3 in mouse embryonic dorsal spinal cord. We found most dILB neuron specific genes previously identified to be directly activated by Tlx3. Surprisingly, we found Tlx3 also directly represses many genes associated with the alternative inhibitory dILA neuronal fate. In both cases, direct targets include transcription factors and terminal differentiation genes, showing that Tlx3 directly controls cell identity at distinct levels. Our findings provide a molecular frame for the master regulatory role of Tlx3 in developing glutamatergic dILB neurons. In addition, they suggest a novel function for Tlx3 as direct repressor of GABAergic dILA identity, pointing to how generation of the two alternative cell fates being tightly coupled.

Foreseeing postoperative pain in neurosurgical patients: pupillometry predicts postoperative pain ratings-an observational study.

Pupillary reflex dilation (PRD) is triggered by noxious stimuli and diminished by opioid administration. In the postoperative period, PRD has been shown to be correlated with pain reporting and a useful tool to guide opioid administration. In this study we assessed whether pupillary measurements taken before extubation were related with the patient's reported pain in the Post-Anesthesia Care Unit (PACU) using the Numerical Rating Scale (NRS). Our objective was to evaluate the correlation of PRD and pupillary variables measured intraoperatively with postoperative pain under the same opioid concentration. This was a prospective observational study of 26 neurosurgical patients undergoing general anesthesia exclusively with propofol and remifentanil. A portable infrared pupillometer was used to provide an objective measure of pupil size and PRD (using the Pupillary Pain Index) before extubation. Pain ratings were obtained from patients after recovery of consciousness, while remifentanil was maintained at 2 ng/mL. A significant correlation was observed between NRS scores and pre-extubation PPI (rS = 0.62; P = 0.002), as well as between NRS scores and pupil diameter before tetanic stimulation PPI (rS = 0.56, P = 0.006). We also found a negative correlation between pupil diameter and age (rS = - 0.42, P = 0.04). The statistically significant correlation between pre-extubation PPI scores and NRS scores, as well as between the pupillary diameter before tetanic stimulation and NRS scores suggest the possibility of titrating analgesia at the end of the intraoperative period based on individual responses. This could allow clinicians to identify the ideal remifentanil concentration for the postoperative period.

Pharmacodynamic modelling of the effect of remifentanil using the Pupillary Pain Index.

Using a targeted controlled infusion of remifentanil during total intravenous anesthesia, we investigated the effect-site concentrations of remifentanil that correlate with different values of the Pupillary Pain Index and which concentrations were necessary for achieving a Pupillary Pain Index ≤ 4 and its usefulness in titrating opioids. The Pupillary Pain Index was measured in 54 patients prior to surgery under different remifentanil effect-site concentrations and subsequently modeled. One hundred and twenty-eight measurements were taken at different remifentanil concentrations while titrating propofol for a similar depth of hypnosis using a BIS monitor. Our modeled Hill equation revealed a remifentanil of 2.96 ng/mL for a PPI of 4, and the probability model a Ce of 3.22 ng/mL for the probability of 50% of patients achieving a PPI score ≤ 4. For the probability of 80% of patients achieving a PPI score ≤ 4 the Ce of remifentanil was 4.39 ng/mL. We conclude that concentrations of remifentanil that have been shown to suppress movement in response to noxious stimulation correspond to a Pupillary Pain Index ≤ 4.

A role for prolyl isomerase PIN1 in the phosphorylation-dependent modulation of PRRXL1 function.

Prrxl1 encodes for a paired-like homeodomain transcription factor essential for the correct establishment of the dorsal root ganglion - spinal cord nociceptive circuitry during development. Prrxl1-null mice display gross anatomical disruption of this circuitry, which translates to a markedly diminished sensitivity to noxious stimuli. Here, by the use of an immunoprecipitation and mass spectrometry approach, we identify five highly conserved phosphorylation sites (T110, S119, S231, S233 and S251) in PRRXL1 primary structure. Four are phospho-S/T-P sites, which suggest a role for the prolyl isomerase PIN1 in regulating PRRXL1. Accordingly, PRRXL1 physically interacts with PIN1 and displays diminished transcriptional activity in a Pin1-null cell line. Additionally, these S/T-P sites seem to be important for PRRXL1 conformation, and their point mutation to alanine or aspartate down-regulates PRRXL1 transcriptional activity. Altogether, our findings provide evidence for a putative novel role of PIN1 in the development of the nociceptive system and indicate phosphorylation-mediated conformational changes as a mechanism for regulating the PRRXL1 role in the process.

Increased fronto-hippocampal connectivity in the Prrxl1 knockout mouse model of congenital hypoalgesia.

Despite the large number of studies addressing how prolonged painful stimulation affects brain functioning, there are only a handful of studies aimed at uncovering if persistent conditions of reduced pain perception would also result in brain plasticity. Permanent hypoalgesia induced by neonatal injection of capsaicin or carrageenan has already been shown to affect learning and memory and to induce alterations in brain gene expression. In this study, we used the Prrxl1 model of congenital mild hypoalgesia to conduct a detailed study of the neurophysiological and behavioral consequences of reduced pain experience. Prrxl1 knockout animals are characterized by selective depletion of small diameter primary afferents and abnormal development of the superficial dorsal laminae of the spinal cord, resulting in diminished pain perception but normal tactile and motor behaviour. Behavioral testing of Prrxl1 mice revealed that these animals have reduced anxiety levels, enhanced memory performance, and improved fear extinction. Neurophysiological recordings from awake behaving Prrxl1 mice show enhanced altered fronto-hippocampal connectivity in the theta- and gamma-bands. Importantly, although inflammatory pain by Complete Freund Adjuvant injection caused a decrease in fronto-hippocampal connectivity in the wild-type animals, Prrxl1 mice maintained the baseline levels. The onset of inflammatory pain also reverted the differences in forebrain expression of stress- and monoamine-related genes in Prrxl1 mice. Altogether our results suggest that congenital hypoalgesia may have an effect on brain plasticity that is the inverse of what is usually observed in animal models of chronic pain.

Zinc finger transcription factor Casz1 expression is regulated by homeodomain transcription factor Prrxl1 in embryonic spinal dorsal horn late-born excitatory interneurons.

The transcription factor Casz1 is required for proper assembly of vertebrate vasculature and heart morphogenesis as well as for temporal control of Drosophila neuroblasts and mouse retina progenitors in the generation of different cell types. Although Casz1 function in the mammalian nervous system remains largely unexplored, Casz1 is expressed in several regions of this system. Here we provide a detailed spatiotemporal characterization of Casz1 expression along mouse dorsal root ganglion (DRG) and dorsal spinal cord development by immunochemistry. In the DRG, Casz1 is broadly expressed in sensory neurons since they are born until perinatal age. In the dorsal spinal cord, Casz1 displays a more dynamic pattern being first expressed in dorsal interneuron 1 (dI1) progenitors and their derived neurons and then in a large subset of embryonic dorsal late-born excitatory (dILB) neurons that narrows gradually to become restricted perinatally to the inner portion. Strikingly, expression analyses using Prrxl1-knockout mice revealed that Prrxl1, a key transcription factor in the differentiation of dILB neurons, is a positive regulator of Casz1 expression in the embryonic dorsal spinal cord but not in the DRG. By performing chromatin immunoprecipitation in the dorsal spinal cord, we identified two Prrxl1-bound regions within Casz1 introns, suggesting that Prrxl1 directly regulates Casz1 transcription. Our work reveals that Casz1 lies downstream of Prrxl1 in the differentiation pathway of a large subset of dILB neurons and provides a framework for further studies of Casz1 in assembly of the DRG-spinal circuit.

Dual role of Tlx3 as modulator of Prrxl1 transcription and phosphorylation.

The proper establishment of the dorsal root ganglion/spinal cord nociceptive circuitry depends on a group of homeodomain transcription factors that includes Prrxl1, Brn3a and Tlx3. By the use of epistatic analysis, it was suggested that Tlx3 and Brn3a, which highly co-localize with Prrxl1 in these tissues, are required to maintain Prrxl1 expression. Here, we report two Tlx3-dependent transcriptional mechanisms acting on Prrxl1 alternative promoters, referred to as P3 and P1/P2 promoters. We demonstrate that (i) Tlx3 induces the transcriptional activity of the TATA-containing promoter P3 by directly binding to a bipartite DNA motif and (ii) it synergistically interacts with Prrxl1 by indirectly activating the Prrxl1 TATA-less promoters P1/P2 via the action of Brn3a. The Tlx3 N-terminal domain 1-38 was shown to have a major role on the overall Tlx3 transcriptional activity and the C-terminus domain (amino acids 256-291) to mediate the Tlx3 effect on promoters P1/P2. On the other hand, the 76-111 domain was shown to decrease Tlx3 activity on the TATA-promoter P3. In addition to its action on Prrxl1 alternative promoters, Tlx3 proved to have the ability to induce Prrxl1 phosphorylation. The Tlx3 domain responsible for Prrxl1 hyperphosphorylation was mapped and encompasses amino acid residues 76 to 111. Altogether, our results suggest that Tlx3 uses distinct mechanisms to tightly modulate Prrxl1 activity, either by controlling its transcriptional levels or by increasing Prrxl1 phosphorylation state.

Paired related homeobox protein-like 1 (Prrxl1) controls its own expression by a transcriptional autorepression mechanism.

The homeodomain factor paired related homeobox protein-like 1 (Prrxl1) is crucial for proper assembly of dorsal root ganglia (DRG)-dorsal spinal cord (SC) pain-sensing circuit. By performing chromatin immunoprecipitation with either embryonic DRG or dorsal SC, we identified two evolutionarily conserved regions (i.e. proximal promoter and intron 4) of Prrxl1 locus that show tissue-specific binding of Prrxl1. Transcriptional assays confirm the identified regions can mediate repression by Prrxl1, while gain-of-function studies in Prrxl1 expressing ND7/23 cells indicate Prrxl1 can down-regulate its own expression. Altogether, our results suggest that Prrxl1 uses distinct regulatory regions to repress its own expression in DRG and dorsal SC.

Ser¹¹9 phosphorylation modulates the activity and conformation of PRRXL1, a homeodomain transcription factor.

PRRXL1 [paired related homeobox-like 1; also known as DRG11 (dorsal root ganglia 11)] is a paired-like homeodomain transcription factor expressed in DRG and dSC (dorsal spinal cord) nociceptive neurons. PRRXL1 is crucial for the establishment and maintenance of nociceptive circuitry, as Prrxl1(-/-) mice present neuronal loss, reduced pain sensitivity and failure to thrive. In the present study, we show that PRRXL1 is highly phosphorylated in vivo, and that its multiple band pattern on electrophoretic analysis is the result of different phosphorylation states. PRRXL1 phosphorylation appears to be differentially regulated along the dSC and DRG development and it is mapped to two functional domains. One region comprises amino acids 107-143, whereas the other one encompasses amino acids 227-263 and displays repressor activity. Using an immunoprecipitation-MS approach, two phosphorylation sites were identified, Ser¹¹9 and Ser²³8. Phosphorylation at Ser¹¹9 is shown to be determinant for PRRXL1 conformation and transcriptional activity. Ser¹¹9 phosphorylation is thus proposed as a mechanism for regulating PRRXL1 function and conformation during nociceptive system development.

The pronociceptive dorsal reticular nucleus contains mostly tonic neurons and shows a high prevalence of spontaneous activity in block preparation.

Despite the importance and significant clinical impact of understanding information processing in the nociceptive system, the functional properties of neurons in many parts of this system are still unknown. In this work we performed whole cell patch-clamp recording in rat brain stem blocks to characterize the electrophysiological properties of neurons in the dorsal reticular nucleus (DRt), a region known to be involved in pronociceptive modulation. We also compared properties of DRt neurons with those in the adjacent parvicellular reticular nucleus and in neighboring regions outside the reticular formation. We found that neurons in the DRt and parvicellular reticular nucleus had similar electrophysiological properties and exhibited mostly toniclike firing patterns, whereas neurons outside the reticular formation showed a larger diversity of firing patterns. Interestingly, more than one-half of the neurons also showed spontaneous activity. While the general view of the reticular formation, being a loosely associated mesh of groups of neurons with diverse function, and earlier reports suggests more electrophysiological heterogeneity, we showed that this is indeed not the case. Our results indicate that functional difference of neurons in the reticular formation may mostly be determined by their connectivity profiles and not by their intrinsic electrophysiological properties. The dominance of tonic neurons in the DRt supports previous conclusions that these neurons encode stimulus intensity through their firing frequency, while the high prevalence of spontaneous activity most likely shapes nociceptive modulation by this brain stem region.

Several cis-regulatory elements control mRNA stability, translation efficiency, and expression pattern of Prrxl1 (paired related homeobox protein-like 1).

The homeodomain transcription factor Prrxl1/DRG11 has emerged as a crucial molecule in the establishment of the pain circuitry, in particular spinal cord targeting of dorsal root ganglia (DRG) axons and differentiation of nociceptive glutamatergic spinal cord neurons. Despite Prrxl1 importance in the establishment of the DRG-spinal nociceptive circuit, the molecular mechanisms that regulate its expression along development remain largely unknown. Here, we show that Prrxl1 transcription is regulated by three alternative promoters (named P1, P2, and P3), which control the expression of three distinct Prrxl1 5'-UTR variants, named 5'-UTR-A, 5'-UTR-B, and 5'-UTR-C. These 5'-UTR sequences confer distinct mRNA stability and translation efficiency to the Prrxl1 transcript. The most conserved promoter (P3) contains a TATA-box and displays in vivo enhancer activity in a pattern that overlaps with the zebrafish Prrxl1 homologue, drgx. Regulatory modules present in this sequence were identified and characterized, including a binding site for Phox2b. Concomitantly, we demonstrate that zebrafish Phox2b is required for the expression of drgx in the facial, glossopharyngeal, and vagal cranial ganglia.

Prefrontal cortex and mediodorsal thalamus reduced connectivity is associated with spatial working memory impairment in rats with inflammatory pain.

The medial prefrontal cortex (mPFC) and the mediodorsal thalamus (MD) form interconnected neural circuits that are important for spatial cognition and memory, but it is not known whether the functional connectivity between these areas is affected by the onset of an animal model of inflammatory pain. To address this issue, we implanted 2 multichannel arrays of electrodes in the mPFC and MD of adult rats and recorded local field potential activity during a food-reinforced spatial working memory task. Recordings were performed for 3weeks, before and after the establishment of the pain model. Our results show that inflammatory pain caused an impairment of spatial working memory performance that is associated with changes in the activity of the mPFC-MD circuit; an analysis of partial directed coherence between the areas revealed a global decrease in the connectivity of the circuit. This decrease was observed over a wide frequency range in both the frontothalamic and thalamofrontal directions of the circuit, but was more evident from MD to mPFC. In addition, spectral analysis revealed significant oscillations of power across frequency bands, namely with a strong theta component that oscillated after the onset of the painful condition. Finally, our data revealed that chronic pain induces an increase in theta/gamma phase coherence and a higher level of mPFC-MD coherence, which is partially conserved across frequency bands. The present results demonstrate that functional disturbances in mPFC-MD connectivity are a relevant cause of deficits in pain-related working memory.

Impaired spatial memory performance in a rat model of neuropathic pain is associated with reduced hippocampus-prefrontal cortex connectivity.

Chronic pain patients commonly complain of working memory deficits, but the mechanisms and brain areas underlying this cognitive impairment remain elusive. The neuronal populations of the mPFC and dorsal CA1 (dCA1) are well known to form an interconnected neural circuit that is crucial for correct performance in spatial memory-dependent tasks. In this study, we investigated whether the functional connectivity between these two areas is affected by the onset of an animal model of peripheral neuropathic pain. To address this issue, we implanted two multichannel arrays of electrodes in the mPFC and dCA1 of rats and recorded the neuronal activity during a food-reinforced spatial working memory task in a reward-based alternate trajectory maze. Recordings were performed for 3 weeks, before and after the establishment of the spared nerve injury model of neuropathy. Our results show that the nerve lesion caused an impairment of working memory performance that is temporally associated with changes in the mPFC populational firing activity patterns when the animals navigated between decision points-when memory retention was most needed. Moreover, the activity of both recorded neuronal populations after the nerve injury increased their phase locking with respect to hippocampal theta rhythm. Finally, our data revealed that chronic pain reduces the overall amount of information flowing in the fronto-hippocampal circuit and induces the emergence of different oscillation patterns that are well correlated with the correct/incorrect performance of the animal on a trial-by-trial basis. The present results demonstrate that functional disturbances in the fronto-hippocampal connectivity are a relevant cause for pain-related working memory deficits.

Inflammatory pain disrupts the orbitofrontal neuronal activity and risk-assessment performance in a rodent decision-making task.

It has been recently described that disruption of the neural mechanisms of emotion-based decision making occurs in both chronic pain patients and in animal models of pain; moreover, it also has been shown that chronic pain causes morphological and functional changes in the prefrontal cortex that may be crucial for this decision-making dysfunction. However, it is not known whether pain alone is capable of altering the neuronal encoding of decision exhibited by prefrontal neurons. We have previously shown that naïve animals have risk-averse performance in the rodent gambling task, whereas chronic pain animals reverse their choice preference and become risk prone. Using this paradigm, we chronically implanted arrays of multielectrodes and recorded from neuronal ensembles in the orbitofrontal cortex of freely moving animals performing 4 sessions of the rodent gambling task: 2 in control conditions and 2 after the onset of inflammatory pain induced by complete Freund's adjuvant injection. Our results show that the instantaneous neuronal firing rate was correlated with the probability of choosing a specific lever in 62.5% of the neurons; however, although in the control sessions 61% of the neurons encoded the reward magnitude, after the pain onset only 16% of the neurons differentiated small from large rewards. Moreover, we found that the fraction of risk-sensitive neurons recorded in each session predicted the overall risk bias of the animal. Our data suggest that orbitofrontal cortex encoding of risk preference is compromised in chronic pain animals.

Dynamics of Circadian Thalamocortical Flow of Information during a Peripheral Neuropathic Pain Condition.

It is known that the thalamocortical loop plays a crucial role in the encoding of sensory-discriminative features of painful stimuli. However, only a few studies have addressed the changes in thalamocortical dynamics that may occur after the onset of chronic pain. Our goal was to evaluate how the induction of chronic neuropathic pain affected the flow of information within the thalamocortical loop throughout the brain states of the sleep-wake cycle. To address this issue we recorded local field potentials (LFPs) - both before and after the establishment of neuropathic pain in awake freely moving adult rats chronically implanted with arrays of multielectrodes in the lateral thalamus and primary somatosensory cortex. Our results show that the neuropathic injury induced changes in the number of wake and slow-wave-sleep (SWS) state episodes, and especially in the total number of transitions between brain states. Moreover, partial directed coherence - analysis revealed that the amount of information flow between cortex and thalamus in neuropathic animals decreased significantly, indicating that the overall thalamic activity had less weight over the cortical activity. However, thalamocortical LFPs displayed higher phase-locking during awake and SWS episodes after the nerve lesion, suggesting faster transmission of relevant information along the thalamocortical loop. The observed changes are in agreement with the hypothesis of thalamic dysfunction after the onset of chronic pain, and may result from diminished inhibitory effect of the primary somatosensory cortex over the lateral thalamus.

Instability of spatial encoding by CA1 hippocampal place cells after peripheral nerve injury.

Several authors have shown that the hippocampus responds to painful stimulation and suggested that prolonged painful conditions could lead to abnormal hippocampal functioning. The aim of the present study was to evaluate whether the induction of persistent peripheral neuropathic pain would affect basic hippocampal processing such as the spatial encoding performed by CA1 place cells. These place cells fire preferentially in a certain spatial position in the environment, and this spatial mapping remains stable across multiple experimental sessions even when the animal is removed from the testing environment. To address the effect of prolonged pain on the stability of place cell encoding, we chronically implanted arrays of electrodes in the CA1 hippocampal region of adult rats and recorded the multichannel neuronal activity during a simple food-reinforced alternation task in a U-shaped runway. The activity of place cells was followed over a 3-week period before and after the establishment of an animal model of neuropathy, spared nerve injury. Our results show that the nerve injury increased the number of place fields encoded per cell and the mapping size of the place fields. In addition, there was an increase in in-field coherence while the amount of spatial information content that a single spike conveyed about the animal location decreased over time. Other measures of spatial tuning (in-field firing rate, firing peak and number of spikes) were unchanged between the experimental groups. These results demonstrate that the functioning of spatial place cells is altered during neuropathic pain conditions.

Pain in portuguese patients with multiple sclerosis.

Early reports often ignored pain as an important symptom in multiple sclerosis (MS). Pain prevalence figures in MS from European countries other than Portugal range between 40 and 65%. To our knowledge there is no published data in English on pain in MS in Portugal. We describe the demographic and clinical characteristics, with an emphasis on pain, of 85 MS patients followed-up in a Portuguese hospital, contributing to pain epidemiology in MS. Patients were interviewed sequentially after their regular appointments at the MS clinic; patients with pain completed The Brief Pain Inventory and The McGill Pain Questionnaire (MPQ). The prevalence of pain found was 34%. Headache and back pain were the most common anatomical sites described, followed by upper and lower limbs. Intensity of pain in an 11-point scale was, for the maximum pain intensity 6.7 ± 1.8, for the minimum pain intensity 2.2 ± 2.0, for the mean pain intensity 4.5 ± 1.5, and for the actual pain intensity 2.4 ± 2.9. Pain interfered significantly with general activity, mood, work, social relations, and enjoyment of life. All MS patients with pain employed words from both the sensory and affective categories of the MPQ to describe it. Patient pain descriptions' included the word "hot-burning" in 59% of the cases, common in the report of central pain, but neuropathic pain medications were only used by 10% of them. Pain is an important symptom in Portuguese patients with MS, not only because of the high prevalence found, concordant with other European countries, but also because of its interference with quality-of-life.

Postnatal expression of the homeobox gene Prrxl1 (Drg11) is increased in inflammatory but not neuropathic pain.

The paired-type homeodomain transcription factor Prrxl1 (also known as Drg11) is a key regulator of the differentiation and survival of dorsal root ganglia (DRG) and spinal nociceptive neurons in pre- and perinatal stages. Prrxl1(-/-) mice exhibit abnormalities in DRG-spinal projections, defects in superficial dorsal horn structure and neurochemistry, and reduced nociceptive behaviour in several pain tests. Although a low expression of Prrxl1 persists in dorsal root ganglia beyond embryonic development, no data exist on its role in adult life. In this paper we evaluate whether DRG expression of Prrxl1 is affected both in inflammatory and neuropathic models of pain in adult mice. Ipsilateral versus contralateral relative expression of Prrxl1 in the DRG was compared in control and pain animals. The expression of Prrxl1 mRNA in mice with zymosan-induced peripheral inflammation presented a 3.06 ± 0.71-fold-increase in ipsilateral ganglia, which was significantly different from the value observed in control animals. In contrast, a slight, non-statistically significant decrease was detected in the SNI model of neuropathy. Interestingly, the expression of the mRNA splice variant Prrxl1b was unchanged in both pain conditions. Immunohistochemical studies showed an increase in the number of Prrxl1-positive neurons in the inflammatory pain model, which belonged both in the peptidergic and non-peptidergic categories. Our present results point to a role for Prrxl1 in sensitization of nociceptive neurons upon inflammatory pain.

Local axon collaterals of lamina I projection neurons in the spinal cord of young rats.

Large, mediolaterally oriented neurons in lamina I of the spinal cord, frequently referred to as marginal cells of Waldeyer, are known to project to supraspinal targets via the anterolateral tract (ALT). Although dendritic organization of lamina I neurons has been extensively studied, little is known about their local axonal morphology and branching. With the help of oblique illumination, we visually identified large lamina I neurons in the isolated lumbar enlargement (L1-L6) of the spinal cord of P14-P20 rats. By using intracellular and cell-attached biocytin injections, we achieved extensive axonal and dendritic labeling in 77 lamina I cells, 40 of which were identified as ALT projection neurons. In the majority of the cases (n = 28), the main axon of these projection neurons gave rise to one or more thin collaterals on the ipsilateral side. Based on their trajectory and location, these collaterals could be divided into three major categories: dorsal, lateral, and ventral. Lamina I projection neurons had dorsal (n = 5), lateral (n = 8), or ventral (n = 6) collaterals only or a combination of these collateral types (n = 9). Our results suggest that lamina I ALT projection neurons can additionally function as local-circuit and propriospinal neurons participating in intra- and intersegmental spinal cord processing.