Inhibitory Interneurons That Express GFP in the PrP-GFP Mouse Spinal Cord Are Morphologically Heterogeneous, Innervated by Several Classes of Primary Afferent and Include Lamina I Projection Neurons among Their Postsynaptic Targets.

The superficial dorsal horn of the spinal cord contains numerous inhibitory interneurons, which regulate the transmission of information perceived as touch, pain, or itch. Despite the importance of these cells, our understanding of their roles in the neuronal circuitry is limited by the difficulty in identifying functional populations. One group that has been identified and characterized consists of cells in the mouse that express green fluorescent protein (GFP) under control of the prion protein (PrP) promoter. Previous reports suggested that PrP-GFP cells belonged to a single morphological class (central cells), received inputs exclusively from unmyelinated primary afferents, and had axons that remained in lamina II. However, we recently reported that the PrP-GFP cells expressed neuronal nitric oxide synthase (nNOS) and/or galanin, and it has been shown that nNOS-expressing cells are more diverse in their morphology and synaptic connections. We therefore used a combined electrophysiological, pharmacological, and anatomical approach to reexamine the PrP-GFP cells. We provide evidence that they are morphologically diverse (corresponding to "unclassified" cells) and receive synaptic input from a variety of primary afferents, with convergence onto individual cells. We also show that their axons project into adjacent laminae and that they target putative projection neurons in lamina I. This indicates that the neuronal circuitry involving PrP-GFP cells is more complex than previously recognized, and suggests that they are likely to have several distinct roles in regulating the flow of somatosensory information through the dorsal horn.

Spinal neurons that contain gastrin-releasing peptide seldom express Fos or phosphorylate extracellular signal-regulated kinases in response to intradermal chloroquine.

BACKGROUND: Gastrin-releasing peptide (GRP) is thought to play a role in the itch evoked by intradermal injection of chloroquine. Although some early studies suggested that GRP was expressed in pruriceptive primary afferents, it is now thought that GRP in the spinal cord is derived mainly from a population of excitatory interneurons in lamina II, and it has been suggested that these are involved in the itch pathway. To test this hypothesis, we used the transcription factor Fos and phosphorylation of extracellular signal-regulated kinases (ERK) to look for evidence that interneurons expressing GRP were activated following intradermal injection of chloroquine into the calf, in mice that express enhanced green fluorescent protein (EGFP) in these cells. RESULTS: Injection of chloroquine resulted in numerous Fos- or phospho-ERK (pERK) positive cells in the somatotopically appropriate part of the superficial dorsal horn. The proportion of all neurons in this region that showed Fos or pERK was 18% and 21%, respectively. However, among the GRP-EGFP, only 7% were Fos-positive and 3% were pERK-positive. As such, GRP-EGFP cells were significantly less likely than other neurons to express Fos or to phosphorylate ERK. CONCLUSIONS: Both expression of Fos and phosphorylation of ERK can be used to identify dorsal horn neurons activated by chloroquine injection. However, these results do not support the hypothesis that interneurons expressing GRP are critical components in the itch pathway.

Presynaptically localized cyclic GMP-dependent protein kinase 1 is a key determinant of spinal synaptic potentiation and pain hypersensitivity.

Synaptic long-term potentiation (LTP) at spinal neurons directly communicating pain-specific inputs from the periphery to the brain has been proposed to serve as a trigger for pain hypersensitivity in pathological states. Previous studies have functionally implicated the NMDA receptor-NO pathway and the downstream second messenger, cGMP, in these processes. Because cGMP can broadly influence diverse ion-channels, kinases, and phosphodiesterases, pre- as well as post-synaptically, the precise identity of cGMP targets mediating spinal LTP, their mechanisms of action, and their locus in the spinal circuitry are still unclear. Here, we found that Protein Kinase G1 (PKG-I) localized presynaptically in nociceptor terminals plays an essential role in the expression of spinal LTP. Using the Cre-lox P system, we generated nociceptor-specific knockout mice lacking PKG-I specifically in presynaptic terminals of nociceptors in the spinal cord, but not in post-synaptic neurons or elsewhere (SNS-PKG-I(-/-) mice). Patch clamp recordings showed that activity-induced LTP at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) was completely abolished in SNS-PKG-I(-/-) mice, although basal synaptic transmission was not affected. Analyses of synaptic failure rates and paired-pulse ratios indicated a role for presynaptic PKG-I in regulating the probability of neurotransmitter release. Inositol 1,4,5-triphosphate receptor 1 and myosin light chain kinase were recruited as key phosphorylation targets of presynaptic PKG-I in nociceptive neurons. Finally, behavioural analyses in vivo showed marked defects in SNS-PKG-I(-/-) mice in several models of activity-induced nociceptive hypersensitivity, and pharmacological studies identified a clear contribution of PKG-I expressed in spinal terminals of nociceptors. Our results thus indicate that presynaptic mechanisms involving an increase in release probability from nociceptors are operational in the expression of synaptic LTP on spinal-PAG projection neurons and that PKG-I localized in presynaptic nociceptor terminals plays an essential role in this process to regulate pain sensitivity.

A putative relay circuit providing low-threshold mechanoreceptive input to lamina I projection neurons via vertical cells in lamina II of the rat dorsal horn.

BACKGROUND: Lamina I projection neurons respond to painful stimuli, and some are also activated by touch or hair movement. Neuropathic pain resulting from peripheral nerve damage is often associated with tactile allodynia (touch-evoked pain), and this may result from increased responsiveness of lamina I projection neurons to non-noxious mechanical stimuli. It is thought that polysynaptic pathways involving excitatory interneurons can transmit tactile inputs to lamina I projection neurons, but that these are normally suppressed by inhibitory interneurons. Vertical cells in lamina II provide a potential route through which tactile stimuli can activate lamina I projection neurons, since their dendrites extend into the region where tactile afferents terminate, while their axons can innervate the projection cells. The aim of this study was to determine whether vertical cell dendrites were contacted by the central terminals of low-threshold mechanoreceptive primary afferents. RESULTS: We initially demonstrated contacts between dendritic spines of vertical cells that had been recorded in spinal cord slices and axonal boutons containing the vesicular glutamate transporter 1 (VGLUT1), which is expressed by myelinated low-threshold mechanoreceptive afferents. To confirm that the VGLUT1 boutons included primary afferents, we then examined vertical cells recorded in rats that had received injections of cholera toxin B subunit (CTb) into the sciatic nerve. We found that over half of the VGLUT1 boutons contacting the vertical cells were CTb-immunoreactive, indicating that they were of primary afferent origin. CONCLUSIONS: These results show that vertical cell dendritic spines are frequently contacted by the central terminals of myelinated low-threshold mechanoreceptive afferents. Since dendritic spines are associated with excitatory synapses, it is likely that most of these contacts were synaptic. Vertical cells in lamina II are therefore a potential route through which tactile afferents can activate lamina I projection neurons, and this pathway could play a role in tactile allodynia.

Projection neurons in lamina III of the rat spinal cord are selectively innervated by local dynorphin-containing excitatory neurons.

Large projection neurons in lamina III of the rat spinal cord that express the neurokinin 1 receptor are densely innervated by peptidergic primary afferent nociceptors and more sparsely by low-threshold myelinated afferents. However, we know little about their input from other glutamatergic neurons. Here we show that these cells receive numerous contacts from nonprimary boutons that express the vesicular glutamate transporter 2 (VGLUT2), and form asymmetrical synapses on their dendrites and cell bodies. These synapses are significantly smaller than those formed by peptidergic afferents, but provide a substantial proportion of the glutamatergic synapses that the cells receive (over a third of those in laminae I-II and half of those in deeper laminae). Surprisingly, although the dynorphin precursor preprodynorphin (PPD) was only present in 4-7% of VGLUT2 boutons in laminae I-IV, it was found in 58% of the VGLUT2 boutons that contacted these cells. This indicates a highly selective targeting of the lamina III projection cells by glutamatergic neurons that express PPD, and these are likely to correspond to local neurons (interneurons and possibly projection cells). Since many PPD-expressing dorsal horn neurons respond to noxious stimulation, this suggests that the lamina III projection cells receive powerful monosynaptic and polysynaptic nociceptive input. Excitatory interneurons in the dorsal horn have been shown to possess I(A) currents, which limit their excitability and can underlie a form of activity-dependent intrinsic plasticity. It is therefore likely that polysynaptic inputs to the lamina III projection neurons are recruited during the development of chronic pain states.

Neurochemical characterisation of lamina II inhibitory interneurons that express GFP in the PrP-GFP mouse.

BACKGROUND: Inhibitory interneurons in the superficial dorsal horn play important roles in modulating sensory transmission, and these roles are thought to be performed by distinct functional populations. We have identified 4 non-overlapping classes among the inhibitory interneurons in the rat, defined by the presence of galanin, neuropeptide Y, neuronal nitric oxide synthase (nNOS) and parvalbumin. The somatostatin receptor sst2A is expressed by ~50% of the inhibitory interneurons in this region, and is particularly associated with nNOS- and galanin-expressing cells. The main aim of the present study was to test whether a genetically-defined population of inhibitory interneurons, those expressing green fluorescent protein (GFP) in the PrP-GFP mouse, belonged to one or more of the neurochemical classes identified in the rat. RESULTS: The expression of sst2A and its relation to other neurochemical markers in the mouse was similar to that in the rat, except that a significant number of cells co-expressed nNOS and galanin. The PrP-GFP cells were entirely contained within the set of inhibitory interneurons that possessed sst2A receptors, and virtually all expressed nNOS and/or galanin. GFP was present in ~3-4% of neurons in the superficial dorsal horn, corresponding to ~16% of the inhibitory interneurons in this region. Consistent with their sst2A-immunoreactivity, all of the GFP cells were hyperpolarised by somatostatin, and this was prevented by administration of a selective sst2 receptor antagonist or a blocker of G-protein-coupled inwardly rectifying K+ channels. CONCLUSIONS: These findings support the view that neurochemistry provides a valuable way of classifying inhibitory interneurons in the superficial laminae. Together with previous evidence that the PrP-GFP cells form a relatively homogeneous population in terms of their physiological properties, they suggest that these neurons have specific roles in processing sensory information in the dorsal horn.

Antibodies Against the Gastrin-releasing Peptide Precursor Pro-Gastrin-releasing Peptide Reveal Its Expression in the Mouse Spinal Dorsal Horn.

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.

Characterisation of NPFF-expressing neurons in the superficial dorsal horn of the mouse spinal cord.

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.

Synaptic circuits involving gastrin-releasing peptide receptor-expressing neurons in the dorsal horn of the mouse spinal cord.

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.

Grpr expression defines a population of superficial dorsal horn vertical cells that have a role in both itch and pain.

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 ∼15% 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.

Neuropeptide Y-expressing dorsal horn inhibitory interneurons gate spinal pain and itch signalling.

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.

Calretinin-expressing islet cells are a source of pre- and post-synaptic inhibition of non-peptidergic nociceptor input to the mouse spinal cord.

Unmyelinated non-peptidergic nociceptors (NP afferents) arborise in lamina II of the spinal cord and receive GABAergic axoaxonic synapses, which mediate presynaptic inhibition. However, until now the source of this axoaxonic synaptic input was not known. Here we provide evidence that it originates from a population of inhibitory calretinin-expressing interneurons (iCRs), which correspond to lamina II islet cells. The NP afferents can be assigned to 3 functionally distinct classes (NP1-3). NP1 afferents have been implicated in pathological pain states, while NP2 and NP3 afferents also function as pruritoceptors. Our findings suggest that all 3 of these afferent types innervate iCRs and receive axoaxonic synapses from them, providing feedback inhibition of NP input. The iCRs also form axodendritic synapses, and their targets include cells that are themselves innervated by the NP afferents, thus allowing for feedforward inhibition. The iCRs are therefore ideally placed to control the input from non-peptidergic nociceptors and pruritoceptors to other dorsal horn neurons, and thus represent a potential therapeutic target for the treatment of chronic pain and itch.

Calretinin-expressing islet cells: a source of pre- and post-synaptic inhibition of non-peptidergic nociceptor input to the mouse spinal cord.

Unmyelinated non-peptidergic nociceptors (NP afferents) arborise in lamina II of the spinal cord and receive GABAergic axoaxonic synapses, which mediate presynaptic inhibition. However, until now the source of this axoaxonic synaptic input was not known. Here we provide evidence that it originates from a population of inhibitory calretinin-expressing interneurons (iCRs), which correspond to lamina II islet cells. The NP afferents can be assigned to 3 functionally distinct classes (NP1-3). NP1 afferents have been implicated in pathological pain states, while NP2 and NP3 afferents also function as pruritoceptors. Our findings suggest that all 3 of these afferent types innervate iCRs and receive axoaxonic synapses from them, providing feedback inhibition of NP input. The iCRs also form axodendritic synapses, and their targets include cells that are themselves innervated by the NP afferents, thus allowing for feedforward inhibition. The iCRs are therefore ideally placed to control the input from non-peptidergic nociceptors and pruritoceptors to other dorsal horn neurons, and thus represent a potential therapeutic target for the treatment of chronic pain and itch.

Sodium-calcium exchanger-3 regulates pain "wind-up": From human psychophysics to spinal mechanisms.

Repeated application of noxious stimuli leads to a progressively increased pain perception; this temporal summation is enhanced in and predictive of clinical pain disorders. Its electrophysiological correlate is "wind-up," in which dorsal horn spinal neurons increase their response to repeated nociceptor stimulation. To understand the genetic basis of temporal summation, we undertook a GWAS of wind-up in healthy human volunteers and found significant association with SLC8A3 encoding sodium-calcium exchanger type 3 (NCX3). NCX3 was expressed in mouse dorsal horn neurons, and mice lacking NCX3 showed normal, acute pain but hypersensitivity to the second phase of the formalin test and chronic constriction injury. Dorsal horn neurons lacking NCX3 showed increased intracellular calcium following repetitive stimulation, slowed calcium clearance, and increased wind-up. Moreover, virally mediated enhanced spinal expression of NCX3 reduced central sensitization. Our study highlights Ca2+ efflux as a pathway underlying temporal summation and persistent pain, which may be amenable to therapeutic targeting.

Galanin-immunoreactivity identifies a distinct population of inhibitory interneurons in laminae I-III of the rat spinal cord.

BACKGROUND: Inhibitory interneurons constitute 30-40% of neurons in laminae I-III and have an important anti-nociceptive role. However, because of the difficulty in classifying them we know little about their organisation. Previous studies have identified 3 non-overlapping groups of inhibitory interneuron, which contain neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin, and have shown that these differ in postsynaptic targets. Some inhibitory interneurons contain galanin and the first aim of this study was to determine whether these form a different population from those containing NPY, nNOS or parvalbumin. We also estimated the proportion of neurons and GABAergic axons that contain galanin in laminae I-III. RESULTS: Galanin cells were concentrated in laminae I-IIo, with few in laminae IIi-III. Galanin showed minimal co-localisation with NPY, nNOS or parvalbumin in laminae I-II, but most galanin-containing cells in lamina III were nNOS-positive. Galanin cells constituted ~7%, 3% and 2% of all neurons in laminae I, II and III, and we estimate that this corresponds to 26%, 10% and 5% of the GABAergic neurons in these laminae. However, galanin was only found in ~6% of GABAergic boutons in laminae I-IIo, and ~1% of those in laminae IIi-III. CONCLUSIONS: These results show that galanin, NPY, nNOS and parvalbumin can be used to define four distinct neurochemical populations of inhibitory interneurons. Together with results of a recent study, they suggest that the galanin and NPY populations account for around half of the inhibitory interneurons in lamina I and a quarter of those in lamina II.

Dynorphin is expressed primarily by GABAergic neurons that contain galanin in the rat dorsal horn.

BACKGROUND: The opioid peptide dynorphin is expressed by certain neurons in the superficial dorsal horn of the spinal cord, but little is known about the types of cell that contain dynorphin. In this study, we have used an antibody against the dynorphin precursor preprodynorphin (PPD), to reveal the cell bodies and axons of dynorphin-expressing neurons in the rat spinal cord. The main aims were to estimate the proportion of neurons in each of laminae I-III that express dynorphin and to determine whether they are excitatory or inhibitory neurons. RESULTS: PPD-immunoreactive cells were concentrated in lamina I and the outer part of lamina II (IIo), where they constituted 17% and 8%, respectively, of all neurons. Around half of those in lamina I and 80% of those in lamina II were GABA-immunoreactive. We have previously identified four non-overlapping neurochemical populations of inhibitory interneurons in this region, defined by the presence of neuropeptide Y, galanin, parvalbumin and neuronal nitric oxide synthase. PPD co-localised extensively with galanin in both cell bodies and axons, but rarely or not at all with the other three markers. PPD was present in around 4% of GABAergic boutons (identified by the presence of the vesicular GABA transporter) in laminae I-II. CONCLUSIONS: These results show that most dynorphin-expressing cells in the superficial dorsal horn are inhibitory interneurons, and that they largely correspond to the population that is defined by the presence of galanin. We estimate that dynorphin is present in ~32% of inhibitory interneurons in lamina I and 11% of those in lamina II. Since the proportion of GABAergic boutons that contain PPD in these laminae was considerably lower than this, our findings suggest that these neurons may generate relatively small axonal arborisations.

Encoding of cutaneous stimuli by lamina I projection neurons.

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.

Characterisation of lamina I anterolateral system neurons that express Cre in a Phox2a-Cre mouse line.

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.

Evidence against AMPA receptor-lacking glutamatergic synapses in the superficial dorsal horn of the rat spinal cord.

Pure NMDA receptor (NMDAr)-mediated EPSCs, thought to correspond to "silent" glutamatergic synapses that lack AMPA receptors (AMPArs), have been observed in superficial spinal dorsal horn of neonatal but not adult rats. Recent anatomical studies suggest that AMPArs are present at virtually all glutamatergic synapses in this region in adults. We used antigen retrieval to examine colocalization of AMPArs and PSD-95 (a marker for glutamatergic synapses) in laminae I-II of neonatal and adult rats. We found a high degree of colocalization in all cases, which suggests that AMPArs are present in the great majority of glutamatergic synapses even in neonatal animals. We therefore reexamined evidence for silent synapses by performing blind whole-cell recordings from superficial dorsal horn neurons in slices from neonatal or adult rats, with focal stimulation to activate glutamatergic synapses. On some occasions in both neonatal (10 of 109, 9%) and adult (9 of 77, 12%) slices, NMDAr-mediated EPSCs were observed when the holding potential was raised to +50 mV at a stimulus strength that had failed to evoke AMPAr-mediated EPSCs. However, in all cases tested, AMPAr-mediated EPSCs were then observed when the cell was returned to -70 mV; this and other properties of the EPSCs suggest that they do not represent genuine silent synapses. When compared with previous findings, our results indicate that the appearance of silent synapses depends on experimental protocol. This suggests that pure NMDAr-mediated EPSCs seen in previous studies do not correspond to AMPAr-lacking synapses but result from another mechanism, for example, loss of labile AMPArs from recently formed synapses.

Substance P-expressing Neurons in the Superficial Dorsal Horn of the Mouse Spinal Cord: Insights into Their Functions and their Roles in Synaptic Circuits.

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.