RESUMEN
The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch, and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here, we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify three clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 and ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.
Asunto(s)
Proteínas de Homeodominio , Animales , Ratones , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismo , Neuronas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Núcleo Celular/metabolismo , Núcleo Celular/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The superficial dorsal horn (SDH) of the spinal cord contains a diverse array of neurons. The vast majority of these are interneurons, most of which are glutamatergic. These can be assigned to several populations, one of which is defined by expression of gastrin-releasing peptide receptor (GRPR). The GRPR cells are thought to be "tertiary pruritoceptors," conveying itch information to lamina I projection neurons of the anterolateral system (ALS). Surprisingly, we recently found that GRPR-expressing neurons belong to a morphological class known as vertical cells, which are believed to transmit nociceptive information to lamina I ALS cells. Little is currently known about synaptic circuits engaged by the GRPR cells. Here we combine viral-mediated expression of PSD95-tagRFP fusion protein with super-resolution microscopy to reveal sources of excitatory input to GRPR cells. We find that they receive a relatively sparse input from peptidergic and non-peptidergic nociceptors in SDH, and a limited input from A- and C-low threshold mechanoreceptors on their ventral dendrites. They receive synapses from several excitatory interneuron populations, including those defined by expression of substance P, neuropeptide FF, cholecystokinin, neurokinin B, and neurotensin. We investigated downstream targets of GRPR cells by chemogenetically exciting them and identifying Fos-positive (activated) cells. In addition to lamina I projection neurons, many ALS cells in lateral lamina V and the lateral spinal nucleus were Fos-positive, suggesting that GRPR-expressing cells target a broader population of projection neurons than was previously recognised. Our findings indicate that GRPR cells receive a diverse synaptic input from various types of primary afferent and excitatory interneuron, and that they can activate ALS cells in both superficial and deep regions of the dorsal horn.
RESUMEN
The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify 3 clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 & ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.
RESUMEN
Somatosensory information is processed by a complex network of interneurons in the spinal dorsal horn. It has been reported that inhibitory interneurons that express neuropeptide Y (NPY), either permanently or during development, suppress mechanical itch, with no effect on pain. Here, we investigate the role of interneurons that continue to express NPY (NPY-INs) in the adult mouse spinal cord. We find that chemogenetic activation of NPY-INs reduces behaviours associated with acute pain and pruritogen-evoked itch, whereas silencing them causes exaggerated itch responses that depend on cells expressing the gastrin-releasing peptide receptor. As predicted by our previous studies, silencing of another population of inhibitory interneurons (those expressing dynorphin) also increases itch, but to a lesser extent. Importantly, NPY-IN activation also reduces behavioural signs of inflammatory and neuropathic pain. These results demonstrate that NPY-INs gate pain and itch transmission at the spinal level, and therefore represent a potential treatment target for pathological pain and itch.
Asunto(s)
Neuralgia , Neuropéptido Y , Ratones , Animales , Neuropéptido Y/genética , Asta Dorsal de la Médula Espinal/patología , Prurito/patología , Interneuronas/fisiología , Médula Espinal/fisiologíaRESUMEN
Excitatory interneurons in the superficial dorsal horn (SDH) are heterogeneous, and include a class known as vertical cells, which convey information to lamina I projection neurons. We recently used pro-NPFF antibody to reveal a discrete population of excitatory interneurons that express neuropeptide FF (NPFF). Here, we generated a new mouse line (NPFFCre) in which Cre is knocked into the Npff locus, and used Cre-dependent viruses and reporter mice to characterise NPFF cell properties. Both viral and reporter strategies labelled many cells in the SDH, and captured most pro-NPFF-immunoreactive neurons (75-80%). However, the majority of labelled cells lacked pro-NPFF, and we found considerable overlap with a population of neurons that express the gastrin-releasing peptide receptor (GRPR). Morphological reconstruction revealed that most pro-NPFF-containing neurons were vertical cells, but these differed from GRPR neurons (which are also vertical cells) in having a far higher dendritic spine density. Electrophysiological recording showed that NPFF cells also differed from GRPR cells in having a higher frequency of miniature EPSCs, being more electrically excitable and responding to a NPY Y1 receptor agonist. Together, these findings indicate that there are at least two distinct classes of vertical cells, which may have differing roles in somatosensory processing.
Asunto(s)
Neuronas , Asta Dorsal de la Médula Espinal , Ratones , Animales , Oligopéptidos , Interneuronas , Receptores de BombesinaRESUMEN
Gastrin-releasing peptide (GRP) in the spinal dorsal horn acts on the GRP receptor, and this signalling mechanism has been strongly implicated in itch. However, the source of GRP in the dorsal horn is not fully understood. For example, the BAC transgenic mouse line GRP::GFP only captures around 25% of GRP-expressing cells, and Grp mRNA is found in several types of excitatory interneuron. A major limitation in attempts to identify GRP-expressing neurons has been that antibodies against GRP cross-react with other neuropeptides, including some that are expressed by primary afferents. Here we have developed two antibodies raised against different parts of the precursor protein, pro-GRP. We show that labelling is specific, and that the antibodies do not cross-react with neuropeptides in primary afferents. Immunoreactivity was strongest in the superficial laminae, and the two antibodies labelled identical structures, including glutamatergic axons and cell bodies. The pattern of pro-GRP-immunoreactivity varied among different neurochemical classes of excitatory interneuron. Cell bodies and axons of all GRP-GFP cells were labelled, confirming reliability of the antibodies. Among the other populations, we found the highest degree of co-expression (>50%) in axons of NPFF-expressing cells, while this was somewhat lower (10-20%) in cells that expressed substance P and NKB, and much lower (<10%) in other classes. Our findings show that these antibodies reliably detect GRP-expressing neurons and axons, and that in addition to the GRP-GFP cells, excitatory interneurons expressing NPFF or substance P are likely to be the main source of GRP in the spinal dorsal horn.
Asunto(s)
Neuropéptidos , Sustancia P , Animales , Ratones , Péptido Liberador de Gastrina/metabolismo , Ratones Transgénicos , Neuropéptidos/metabolismo , Células del Asta Posterior/metabolismo , Reproducibilidad de los Resultados , Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Sustancia P/metabolismoRESUMEN
ABSTRACT: Neurons in the superficial dorsal horn that express the gastrin-releasing peptide receptor (GRPR) are strongly implicated in spinal itch pathways. However, a recent study reported that many of these correspond to vertical cells, a population of interneurons that are believed to transmit nociceptive information. In this study, we have used a GRPR CreERT2 mouse line to identify and target cells that possess Grpr mRNA. We find that the GRPR cells are highly concentrated in lamina I and the outer part of lamina II, that they are all glutamatergic, and that they account for â¼15% of the excitatory neurons in the superficial dorsal horn. We had previously identified 6 neurochemically distinct excitatory interneuron populations in this region based on neuropeptide expression and the GRPR cells are largely separate from these, although they show some overlap with cells that express substance P. Anatomical analysis revealed that the GRPR neurons are indeed vertical cells, and that their axons target each other, as well as arborising in regions that contain projection neurons: lamina I, the lateral spinal nucleus, and the lateral part of lamina V. Surprisingly, given the proposed role of GRPR cells in itch, we found that most of the cells received monosynaptic input from Trpv1-expressing (nociceptive) afferents, that the majority responded to noxious and pruritic stimuli, and that chemogenetically activating them resulted in pain-related and itch-related behaviours. Together, these findings suggest that the GRPR cells are involved in spinal cord circuits that underlie both pain and itch.
Asunto(s)
Células del Asta Posterior , Receptores de Bombesina , Ratones , Animales , Receptores de Bombesina/genética , Receptores de Bombesina/metabolismo , Péptido Liberador de Gastrina/genética , Péptido Liberador de Gastrina/metabolismo , Células del Asta Posterior/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Interneuronas/metabolismo , Prurito/metabolismo , Dolor/metabolismoRESUMEN
Projection neurons belonging to the anterolateral system (ALS) underlie the perception of pain, skin temperature and itch. Many ALS cells are located in laminae III-V of the dorsal horn and the adjacent lateral white matter. However, relatively little is known about the excitatory synaptic input to these deep ALS cells, and therefore about their engagement with the neuronal circuitry of the region. We have used a recently developed mouse line, Phox2a::Cre, to investigate a population of deep dorsal horn ALS neurons known as "antenna cells", which are characterised by dense innervation from peptidergic nociceptors, and to compare these with other ALS cells in the deep dorsal horn and lateral white matter. We show that these two classes differ, both in the density of excitatory synapses, and in the source of input at these synapses. Peptidergic nociceptors account for around two-thirds of the excitatory synapses on the antenna cells, but for only a small proportion of the input to the non-antenna cells. Conversely, boutons with high levels of VGLUT2, which are likely to originate mainly from glutamatergic spinal neurons, account for only â¼5% of the excitatory synapses on antenna cells, but for a much larger proportion of the input to the non-antenna cells. VGLUT1 is expressed by myelinated low-threshold mechanoreceptors and corticospinal axons, and these innervate both antenna and non-antenna cells. However, the density of VGLUT1 input to the non-antenna cells is highly variable, consistent with the view that these neurons are functionally heterogeneous.
Asunto(s)
Esclerosis Amiotrófica Lateral , Animales , Proteínas de Homeodominio/genética , Integrasas , Ratones , Neuronas/fisiología , Células del Asta Posterior/fisiología , Médula Espinal , Asta Dorsal de la Médula EspinalRESUMEN
Mechanical allodynia (pain to normally innocuous tactile stimuli) is a widespread symptom of inflammatory and neuropathic pain. Spinal or medullary dorsal horn (SDH or MDH) circuits mediating tactile sensation and pain need to interact in order to evoke mechanical allodynia. PKCγ-expressing (PKCγ+) interneurons and inhibitory controls within SDH/MDH inner lamina II (IIi) are pivotal in connecting touch and pain circuits. However, the relative contribution of GABA and glycine to PKCγ+ interneuron inhibition remains unknown. We characterized inhibitory inputs onto PKCγ+ interneurons by combining electrophysiology to record spontaneous and miniature IPSCs (sIPSCs, mIPSCs) and immunohistochemical detection of GABAARα2 and GlyRα1 subunits in adult rat MDH. While GlyR-only- and GABAAR-only-mediated mIPSCs/sIPSCs are predominantly recorded from PKCγ+ interneurons, immunohistochemistry reveals that ~80% of their inhibitory synapses possess both GABAARα2 and GlyRα1. Moreover, nearly all inhibitory boutons at gephyrin-expressing synapses on these cells contain glutamate decarboxylase and are therefore GABAergic, with around half possessing the neuronal glycine transporter (GlyT2) and therefore being glycinergic. Thus, while GABA and glycine are presumably co-released and GABAARs and GlyRs are present at most inhibitory synapses on PKCγ+ interneurons, these interneurons exhibit almost exclusively GABAAR-only and GlyR-only quantal postsynaptic inhibitory currents, suggesting a pharmacological specialization of their inhibitory synapses.
Asunto(s)
Hiperalgesia , Receptores de Glicina , Animales , Glicina/farmacología , Interneuronas/metabolismo , Dolor , Ratas , Receptores de Glicina/metabolismo , Sustancia Gelatinosa/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-AminobutíricoRESUMEN
A recently developed Phox2a::Cre mouse line has been shown to capture anterolateral system (ALS) projection neurons. Here, we used this line to test whether Phox2a-positive cells represent a distinct subpopulation among lamina I ALS neurons. We show that virtually all lamina I Phox2a cells can be retrogradely labelled from injections targeted on the lateral parabrachial area (LPb), and that most of those in the cervical cord also belong to the spinothalamic tract. Phox2a cells accounted for ~ 50-60% of the lamina I cells retrogradely labelled from LPb or thalamus. Phox2a was preferentially associated with smaller ALS neurons, and with those showing relatively weak neurokinin 1 receptor expression. The Phox2a cells were also less likely to project to the ipsilateral LPb. Although most Phox2a cells phosphorylated extracellular signal-regulated kinases following noxious heat stimulation, ~ 20% did not, and these were significantly smaller than the activated cells. This suggests that those ALS neurons that respond selectively to skin cooling, which have small cell bodies, may be included among the Phox2a population. Previous studies have defined neurochemical populations among the ALS cells, based on expression of Tac1 or Gpr83. However, we found that the proportions of Phox2a cells that expressed these genes were similar to the proportions reported for all lamina I ALS neurons, suggesting that Phox2a is not differentially expressed among cells belonging to these populations. Finally, we used a mouse line that resulted in membrane labelling of the Phox2a cells and showed that they all possess dendritic spines, although at a relatively low density. However, the distribution of the postsynaptic protein Homer revealed that dendritic spines accounted for a minority of the excitatory synapses on these cells. Our results confirm that Phox2a-positive cells in lamina I are ALS neurons, but show that the Phox2a::Cre line preferentially captures specific types of ALS cells.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Neuronas , Asta Dorsal de la Médula Espinal , Animales , Ratones , Ratones Transgénicos , Neuronas/citología , Neuronas/metabolismo , Asta Dorsal de la Médula Espinal/citología , Asta Dorsal de la Médula Espinal/metabolismo , Sinapsis , Tálamo/citologíaRESUMEN
ABSTRACT: Lamina I of the dorsal horn, together with its main output pathway, lamina I projection neurons, has long been implicated in the processing of nociceptive stimuli, as well as the development of chronic pain conditions. However, the study of lamina I projection neurons is hampered by technical challenges, including the low throughput and selection biases of traditional electrophysiological techniques. Here we report on a technique that uses anatomical labelling strategies and in vivo imaging to simultaneously study a network of lamina I projection neurons in response to electrical and natural stimuli. Although we were able to confirm the nociceptive involvement of this group of cells, we also describe an unexpected preference for innocuous cooling stimuli. We were able to characterize the thermal responsiveness of these cells in detail and found cooling responses decline when exposed to stable cold temperatures maintained for more than a few seconds, as well as to encode the intensity of the end temperature, while heating responses showed an unexpected reliance on adaptation temperatures.
Asunto(s)
Piel , Asta Dorsal de la Médula Espinal , Frío , Interneuronas , Médula EspinalRESUMEN
Dorsal horn excitatory interneurons that express gastrin-releasing peptide (GRP) are part of the circuit for pruritogen-evoked itch. They have been extensively studied in a transgenic line in which enhanced green fluorescent protein (eGFP) is expressed under control of the Grp gene. The GRP-eGFP cells are separate from several other neurochemically-defined excitatory interneuron populations, and correspond to a class previously defined as transient central cells. However, mRNA for GRP is widely distributed among excitatory interneurons in superficial dorsal horn. Here we show that although Grp mRNA is present in several transcriptomically-defined populations, eGFP is restricted to a discrete subset of cells in the GRP::eGFP mouse, some of which express the neuromedin receptor 2 and likely belong to a cluster defined as Glut8. We show that these cells receive much of their excitatory synaptic input from MrgA3/MrgD-expressing nociceptive/pruritoceptive afferents and C-low threshold mechanoreceptors. Although the cells were not innervated by pruritoceptors expressing brain natriuretic peptide (BNP) most of them contained mRNA for NPR1, the receptor for BNP. In contrast, these cells received only ~ 10% of their excitatory input from other interneurons. These findings demonstrate that the GRP-eGFP cells constitute a discrete population of excitatory interneurons with a characteristic pattern of synaptic input.
Asunto(s)
Proteínas Fluorescentes Verdes/genética , Interneuronas/citología , Interneuronas/metabolismo , Sustancia Gelatinosa/metabolismo , Animales , Expresión Génica , Ratones , Sinapsis/metabolismoRESUMEN
The tachykinin peptide substance P (SP) is expressed by many interneurons and some projection neurons in the superficial dorsal horn of the spinal cord. We have recently shown that SP-expressing excitatory interneurons in lamina II correspond largely to a morphological class known as radial cells. However, little is known about their function, or their synaptic connectivity. Here we use a modification of the Brainbow technique to define the excitatory synaptic input to SP radial cells. We show that around half of their excitatory synapses (identified by expression of Homer) are from boutons with VGLUT2, which are likely to originate mainly from local interneurons. The remaining synapses presumably include primary afferents, which generally have very low levels of VGLUT2. Our results also suggest that the SP cells are preferentially innervated by a population of excitatory interneurons defined by expression of green fluorescent protein under control of the gene for gastrin-releasing peptide, and that they receive sparser input from other types of excitatory interneuron. We show that around 40% of lamina I projection neurons express Tac1, the gene encoding substance P. Finally, we show that silencing Tac1-expressing cells in the dorsal horn results in a significant reduction in reflex responses to cold and radiant heat, but does not affect withdrawal to von Frey hairs, or chloroquine-evoked itch.
Asunto(s)
Asta Dorsal de la Médula Espinal , Sustancia P , Animales , Péptido Liberador de Gastrina , Interneuronas , Ratones , Neuronas , Células del Asta Posterior , Médula EspinalRESUMEN
The great majority of neurons in the superficial dorsal horn of the spinal cord are excitatory interneurons, and these are required for the normal perception of pain and itch. We have previously identified 5 largely non-overlapping populations among these cells, based on the expression of four different neuropeptides (cholecystokinin, neurotensin, neurokinin B and substance P) and of green fluorescent protein driven by the promoter for gastrin-releasing peptide (GRP) in a transgenic mouse line. Another peptide (neuropeptide FF, NPFF) has been identified among the excitatory neurons, and here we have used an antibody against the NPFF precursor (pro-NPFF) and a probe that recognises Npff mRNA to identify and characterise these cells. We show that they are all excitatory interneurons, and are separate from the five populations listed above, accounting for ~6% of the excitatory neurons in laminae I-II. By examining phosphorylation of extracellular signal-regulated kinases, we show that the NPFF cells can respond to different types of noxious and pruritic stimulus. Ablation of somatostatin-expressing dorsal horn neurons has been shown to result in a dramatic reduction in mechanical pain sensitivity, while somatostatin released from these neurons is thought to contribute to itch. Since the great majority of the NPFF cells co-expressed somatostatin, these cells may play a role in the perception of pain and itch.
Asunto(s)
Interneuronas/efectos de los fármacos , Oligopéptidos/farmacología , Células del Asta Posterior/efectos de los fármacos , Asta Dorsal de la Médula Espinal/efectos de los fármacos , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Péptido Liberador de Gastrina/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Interneuronas/metabolismo , Ratones Transgénicos , Neuronas/metabolismo , Neurotensina/metabolismo , Células del Asta Posterior/metabolismo , Médula Espinal/metabolismo , Médula Espinal/fisiopatología , Asta Dorsal de la Médula Espinal/metabolismoRESUMEN
Excitatory interneurons account for the majority of dorsal horn neurons, and are required for perception of normal and pathological pain. We have identified largely non-overlapping populations in laminae I-III, based on expression of substance P, gastrin-releasing peptide, neurokinin B, and neurotensin. Cholecystokinin (CCK) is expressed by many dorsal horn neurons, particularly in the deeper laminae. Here, we have used immunocytochemistry and in situ hybridization to characterize the CCK cells. We show that they account for ~7% of excitatory neurons in laminae I-II, but between a third and a quarter of those in lamina III. They are largely separate from the neurokinin B, neurotensin, and gastrin-releasing peptide populations, but show limited overlap with the substance P cells. Laminae II-III neurons with protein kinase Cγ (PKCγ) have been implicated in mechanical allodynia following nerve injury, and we found that around 50% of CCK cells were PKCγ-immunoreactive. Neurotensin is also expressed by PKCγ cells, and among neurons with moderate to high levels of PKCγ, ~85% expressed CCK or neurotensin. A recent transcriptomic study identified mRNA for thyrotropin-releasing hormone in a specific subpopulation of CCK neurons, and we show that these account for half of the CCK/PKCγ cells. These findings indicate that the CCK cells are distinct from other excitatory interneuron populations that we have defined. They also show that PKCγ cells can be assigned to different classes based on neuropeptide expression, and it will be important to determine the differential contribution of these classes to neuropathic allodynia.
Asunto(s)
Colecistoquinina/metabolismo , Interneuronas/citología , Interneuronas/metabolismo , Células del Asta Posterior/citología , Células del Asta Posterior/metabolismo , Animales , Colecistoquinina/análisis , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Around 75% of neurons in laminae I-II of the mouse dorsal horn are excitatory interneurons, and these are required for normal pain perception. We have shown that four largely non-overlapping excitatory interneuron populations can be defined by expression of the neuropeptides neurotensin, neurokinin B (NKB), gastrin-releasing peptide (GRP) and substance P. In addition, we recently identified a population of excitatory interneurons in glabrous skin territory that express dynorphin. The calcium-binding protein calretinin is present in many excitatory neurons in this region, but we know little about its relation to these neuropeptide markers. Here we show that calretinin is differentially expressed, being present in the majority of substance P-, GRP- and NKB-expressing cells, but not in the neurotensin or dynorphin cells. Calretinin-positive cells have been implicated in detection of noxious mechanical stimuli, but are not required for tactile allodynia after neuropathic pain. Our findings are therefore consistent with the suggestion that neuropathic allodynia involves the neurotensin and/or dynorphin excitatory interneuron populations. Around a quarter of inhibitory interneurons in lamina I-II contain calretinin, and recent transcriptomic studies suggest that these co-express substance P. We confirm this, by showing that inhibitory Cre-expressing cells in a Tac1Cre knock-in mouse are calretinin-immunoreactive. Interestingly, there is evidence that these cells express low levels of peptidylglycine alpha-amidating monooxygenase, an enzyme required for maturation of neuropeptides. This may explain our previous finding that although the substance P precursor preprotachykinin A can be detected in some inhibitory interneurons, very few inhibitory axonal boutons are immunoreactive for substance P.
Asunto(s)
Calbindina 2/metabolismo , Interneuronas/metabolismo , Médula Espinal/metabolismo , Animales , Expresión Génica , Inmunohistoquímica , Interneuronas/citología , Masculino , Ratones Transgénicos , Microscopía Confocal , Médula Espinal/citologíaRESUMEN
Excitatory interneurons account for the majority of neurons in the superficial dorsal horn, but despite their presumed contribution to pain and itch, there is still limited information about their organisation and function. We recently identified 2 populations of excitatory interneuron defined by expression of gastrin-releasing peptide (GRP) or substance P (SP). Here, we demonstrate that these cells show major differences in their morphological, electrophysiological, and pharmacological properties. Based on their somatodendritic morphology and firing patterns, we propose that the SP cells correspond to radial cells, which generally show delayed firing. By contrast, most GRP cells show transient or single-spike firing, and many are likely to correspond to the so-called transient central cells. Unlike the SP cells, few of the GRP cells had long propriospinal projections, suggesting that they are involved primarily in local processing. The 2 populations also differed in responses to neuromodulators, with most SP cells, but few GRP cells, responding to noradrenaline and 5-HT; the converse was true for responses to the µ-opioid agonist DAMGO. Although a recent study suggested that GRP cells are innervated by nociceptors and are strongly activated by noxious stimuli, we found that very few GRP cells receive direct synaptic input from TRPV1-expressing afferents, and that they seldom phosphorylate extracellular signal-regulated kinases in response to noxious stimuli. These findings indicate that the SP and GRP cells differentially process somatosensory information.
Asunto(s)
Péptido Liberador de Gastrina/metabolismo , Interneuronas/fisiología , Asta Dorsal de la Médula Espinal/citología , Sustancia P/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Analgésicos/farmacología , Animales , Capsaicina/farmacología , Toxina del Cólera/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Péptido Liberador de Gastrina/genética , Técnicas In Vitro , Interneuronas/efectos de los fármacos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurotransmisores/farmacología , Técnicas de Placa-Clamp , Estimulación Física , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , ARN Mensajero/metabolismo , Fármacos del Sistema Sensorial/farmacología , Estadísticas no Paramétricas , Sustancia P/genética , Taquicininas/genética , Taquicininas/metabolismo , Transducción GenéticaRESUMEN
The superficial dorsal horn (laminae I and II) of the spinal cord contains numerous excitatory and inhibitory interneurons, and recent studies have shown that each of these groups can be divided into several neurochemically distinct populations. Although it has long been known that some neurons in this region have intersegmental (propriospinal) axonal projections, there have been conflicting reports concerning the number of propriospinal cells and the extent of their axons. In addition, little is known about the neurochemical phenotype of propriospinal neurons or about the termination pattern of their axons. In the present study we show, using retrograde tracing, that around a third of lamina I-II neurons in the lumbar enlargement project at least five segments cranially. Substance P-expressing excitatory neurons are over-represented among these cells, accounting for one-third of the propriospinal neurons. In contrast, inhibitory interneurons and excitatory PKCγ neurons are both under-represented among the retrogradely labelled cells. By combining viral vector-mediated Cre-dependent anterograde tracing with immunocytochemistry, we provide evidence that the lateral spinal nucleus (LSN), rather than the superficial dorsal horn, is the main target for axons belonging to propriospinal substance P-expressing neurons. These findings help to resolve the discrepancies between earlier studies and have implications for the role of the LSN in pain mechanisms.
Asunto(s)
Interneuronas/fisiología , Asta Dorsal de la Médula Espinal/citología , Sustancia P/metabolismo , Animales , Toxina del Cólera/metabolismo , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Red Nerviosa/metabolismo , Factor de Transcripción PAX2/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Proteína Quinasa C/metabolismo , Transducción GenéticaRESUMEN
Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2-/-) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2-/- mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability.
Asunto(s)
Ganglios Espinales/fisiopatología , Inmunoglobulina G/administración & dosificación , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Dolor Nociceptivo/inmunología , Dolor Nociceptivo/fisiopatología , Células Receptoras Sensoriales/fisiología , Animales , Células Cultivadas , Femenino , Humanos , Inmunización Pasiva , Masculino , Mecanotransducción Celular , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Células del Asta Posterior/fisiología , Canales de Potasio de la Superfamilia Shaker/fisiologíaRESUMEN
Remyelination occurs in demyelinated lesions in multiple sclerosis (MS) and pharmacological treatments that enhance this process will critically impact the long term functional outcome in the disease. Sildenafil, a cyclic GMP (cGMP)-specific phosphodiesterase 5 inhibitor (PDE5-I), is an oral vasodilator drug extensively used in humans for treatment of erectile dysfunction and pulmonary arterial hypertension. PDE5 is expressed in central nervous system (CNS) neuronal and glial populations and in endothelial cells and numerous studies in rodent models of neurological disease have evidenced the neuroprotective potential of PDE5-Is. Using myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) as a MS model, we previously showed that daily administration of sildenafil starting at peak disease rapidly ameliorates clinical symptoms while administration at symptoms onset prevents disease progression. These beneficial effects of the drug involved down-regulation of adaptive and innate immune responses, protection of axons and oligodendrocytes (OLs) and promotion of remyelination. In this work we have investigated mechanisms involved in the remyelinating effect of sildenafil. Using demyelinated organotypic cerebellar slice cultures we demonstrate that sildenafil stimulates remyelination by direct effects on CNS cells in a nitric oxide (NO)-cGMP-protein kinase G (PKG)-dependent manner. We also show that sildenafil treatment enhances OL maturation and induces expression of the promyelinating factor ciliary neurotrophic factor (CNTF) in spinal cord of EAE mice and in cerebellar slice cultures. Furthermore, we demonstrate that sildenafil promotes a M2 phenotype in bone marrow derived macrophages (BMDM) and increases myelin phagocytosis in these cells and in M2 microglia/macrophages in the spinal cord of EAE mice. Taken together these data indicate that promotion of OL maturation directly or through induction of growth factor expression, regulation of microglia/macrophage inflammatory phenotype and clearance of myelin debris may be relevant mechanisms involved in sildenafil enhancement of remyelination in demyelinated tissue and further support the contention that this well tolerated drug could be useful for ameliorating MS pathology.