Mouse Superficial Dorsal Horn Research Articles

Influence of Early-Life Stress on the Excitability of Dynorphin Neurons in the Adult Mouse Dorsal Horn

While early-life adversity has been associated with a higher risk of developing chronic pain in adulthood, the cellular and molecular mechanisms by which chronic stress during the neonatal period can persistently sensitize developing nociceptive circuits remain poorly understood. Here, we investigate the effects of early-life stress (ELS) on synaptic integration and intrinsic excitability in dynorphin-lineage (DYN) interneurons within the adult mouse superficial dorsal horn (SDH), which are important for inhibiting mechanical pain and itch. The administration of neonatal limited bedding between postnatal days (P)2 and P9 evoked sex-dependent effects on spontaneous glutamatergic signaling, as female SDH neurons exhibited a higher amplitude of miniature excitatory postsynaptic currents (mEPSCs) after ELS, while mEPSC frequency was reduced in DYN neurons of the male SDH. Furthermore, ELS decreased the frequency of miniature inhibitory postsynaptic currents selectively in female DYN neurons. As a result, ELS increased the balance of spontaneous excitation versus inhibition (E:I ratio) in mature DYN neurons of the female, but not male, SDH network. Nonetheless, ELS weakened the total primary afferent-evoked glutamatergic drive onto adult DYN neurons selectively in females, without modifying afferent-evoked inhibitory signaling onto the DYN population. Finally, ELS failed to significantly change the intrinsic membrane excitability of mature DYN neurons in either males or females. Collectively, these data suggest that ELS exerts a long-term influence on the properties of synaptic transmission onto DYN neurons within the adult SDH, which includes a reduction in the overall strength of sensory input onto this important subset of inhibitory interneurons. PerspectiveThis study suggests that chronic stress during the neonatal period influences synaptic function within adult spinal nociceptive circuits in a sex-dependent manner. These findings yield new insight into the potential mechanisms by which early-life adversity might shape the maturation of pain pathways in the central nervous system (CNS).

Effects of Corticosterone on the Excitability of Glutamatergic and GABAergic Neurons of the Adolescent Mouse Superficial Dorsal Horn.

Stress evokes age-dependent effects on pain sensitivity and commonly occurs during adolescence. However, the mechanisms linking adolescent stress and pain remain poorly understood, in part due to a lack of information regarding how stress hormones modulate the function of nociceptive circuits in the adolescent CNS. Here we investigate the short- and long-term effects of corticosterone (CORT) on the excitability of GABAergic and presumed glutamatergic neurons of the spinal superficial dorsal horn (SDH) in Gad1-GFP mice at postnatal days (P)21-P34. In situ hybridization revealed that glutamatergic SDH neurons expressed significantly higher mRNA levels of both glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) compared to adjacent GABAergic neurons. The incubation of spinal cord slices with CORT (90min) evoked select long-term changes in spontaneous synaptic transmission across both cell types in a sex-dependent manner, without altering the intrinsic firing of either Gad1-GFP+ or GFP- neurons. Meanwhile, the acute bath application of CORT significantly decreased the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs), as well as the frequency of miniature inhibitory postsynaptic currents (mIPSCs), in both cell types leading to a net reduction in the balance of spontaneous excitation vs. inhibition (E:I ratio). This CORT-induced reduction in the E:I ratio was not prevented by selective antagonists of either GR (mifepristone) or MR (eplerenone), although eplerenone blocked the effect on mEPSC amplitude. Collectively, these data suggest that corticosterone modulates synaptic function within the adolescent SDH which could influence the overall excitability and output of the spinal nociceptive network.

Short-Term Effects of Corticosterone on the Excitability of the Mouse Superficial Dorsal Horn

Mounting preclinical and clinical evidence demonstrate that the behavioral and cellular consequences of stress vary across different stages of development. Cortisol, or corticosterone (CORT) in rodents, is a major stress hormone and which can rapidly modulate neuronal excitability in multiple brain regions. However, its short-term effects on the function of nociceptive circuits in the developing spinal superficial dorsal horn (SDH) are poorly understood. Here we examined the expression of glucocorticoid receptors (GR) in the adolescent SDH and explored the effects of CORT on intrinsic membrane excitability and synaptic transmission in GABAergic and presumed glutamatergic SDH neurons using Glutamic acid decarboxylase 67-Enhanced Green Fluorescent Protein (Gad67-EGFP) mice. In situ hybridization revealed that Gad67-EGFP-negative SDH neurons exhibited a significantly higher number of GR mRNA puncta compared to adjacent Gad67-EGFP-positive SDH neurons. In vitro patch clamp recordings from spinal cord slices suggested that the acute application of CORT (100 nM) reduced glutamate or GABA release onto a subset of SDH neurons regardless of cell type. However, incubation of spinal cord slices with CORT for 90 minutes failed to influence the intrinsic membrane properties (including rheobase, resting potential and repetitive firing frequency) or miniature postsynaptic currents (mPSCs) in either neuronal population compared to the vehicle control. Collectively, while these results suggest that GR activation fails to exert intermediate effects on the excitability of the adolescent SDH network, the abundance of GR expression in the region raises the possibility that CORT can evoke slow changes in the excitability of dorsal horn neurons via genomic mechanisms. Grant support from NS072202.

Neonatal Injury Alters Sensory Input and Synaptic Plasticity in GABAergic Interneurons of the Adult Mouse Dorsal Horn.

Neonatal tissue injury disrupts the balance between primary afferent-evoked excitation and inhibition onto adult spinal projection neurons. However, whether this reflects cell-type-specific alterations at synapses onto ascending projection neurons, or rather is indicative of global changes in synaptic signaling across the mature superficial dorsal horn (SDH), remains unknown. Therefore the present study investigated the effects of neonatal surgical injury on primary afferent synaptic input to adult mouse SDH interneurons using in vitro patch-clamp techniques. Hindpaw incision at postnatal day (P)3 significantly diminished total primary afferent-evoked glutamatergic drive to adult Gad67-GFP and non-GFP neurons, and reduced their firing in response to sensory input, in both males and females. Early tissue damage also shaped the relative prevalence of monosynaptic A- versus C-fiber-mediated input to mature GABAergic neurons, with an increased prevalence of Aβ- and Aδ-fiber input observed in neonatally-incised mice compared with naive littermate controls. Paired presynaptic and postsynaptic stimulation at an interval that exclusively produced spike timing-dependent long-term potentiation (t-LTP) in projection neurons predominantly evoked NMDAR-dependent long-term depression in naive Gad67-GFP interneurons. Meanwhile, P3 tissue damage enhanced the likelihood of t-LTP generation at sensory synapses onto the mature GABAergic population, and increased the contribution of Ca2+-permeable AMPARs to the overall glutamatergic response. Collectively, the results indicate that neonatal injury suppresses sensory drive to multiple subpopulations of interneurons in the adult SDH, which likely represents one mechanism contributing to reduced feedforward inhibition of ascending projection neurons, and the priming of developing pain pathways, following early life trauma.SIGNIFICANCE STATEMENT Mounting clinical and preclinical evidence suggests that neonatal tissue damage can result in long-term changes in nociceptive processing within the CNS. Although recent work has demonstrated that early life injury weakens the ability of sensory afferents to evoke feedforward inhibition of adult spinal projection neurons, the underlying circuit mechanisms remain poorly understood. Here we demonstrate that neonatal surgical injury leads to persistent deficits in primary afferent drive to both GABAergic and presumed glutamatergic neurons in the mature superficial dorsal horn (SDH), and modifies activity-dependent plasticity at sensory synapses onto the GABAergic population. The functional denervation of spinal interneurons within the mature SDH may contribute to the "priming" of developing pain pathways following early life injury.

Origin and classification of spontaneous discharges in mouse superficial dorsal horn neurons

Superficial laminae of the spinal cord possess a considerable number of neurons with spontaneous activity as reported in vivo and in vitro preparations of several species. Such neurons may play a role in the development of the nociceptive system and/or in the spinal coding of somatosensory signals. We have used electrophysiological techniques in a horizontal spinal cord slice preparation from adult mice to investigate how this activity is generated and what are the main patterns of activity that can be found. The results show the existence of neurons that fire regularly and irregularly. Within each of these main types, it was possible to distinguish patterns of spontaneous activity formed by single action potentials and different types of bursts according to intra-burst firing frequency. Activity in neurons with irregular patterns was blocked by a mixture of antagonists of the main neurotransmitter receptors present in the cord. Approximately 82% of neurons with a regular firing pattern were insensitive to synaptic antagonists but their activity was inhibited by specific ion channel blockers. It is suggested that these neurons generate endogenous activity due to the functional expression of hyperpolarisation-activated and persistent sodium currents driving the activity of irregular neurons.

A quantitative study of neurochemically defined populations of inhibitory interneurons in the superficial dorsal horn of the mouse spinal cord

Around a quarter of neurons in laminae I–II of the dorsal horn are inhibitory interneurons. These play an important role in modulating somatosensory information, including that perceived as pain or itch. Previous studies in rat identified four largely non-overlapping neurochemical populations among these cells, defined by expression of galanin, neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS) or parvalbumin. The galanin cells were subsequently shown to coexpress dynorphin. Several recent studies have used genetically modified mice to investigate the function of different interneuron populations, and it is therefore important to determine whether the same pattern applies in mouse, and to estimate the relative sizes of these populations. We show that the neurochemical organization of inhibitory interneurons in mouse superficial dorsal horn is similar to that in the rat, although a larger proportion of these neurons (33%) express NPY. Between them, these four populations account for ∼75% of inhibitory cells in laminae I–II. Since ∼25% of inhibitory interneurons in this region belong to a novel calretinin-expressing type, our results suggest that virtually all inhibitory interneurons in superficial dorsal horn can be assigned to one of these five neurochemical populations. Although our main focus was inhibitory neurons, we also identified a population of excitatory dynorphin-expressing cells in laminae I–II that are largely restricted to the medial part of the mid-lumbar dorsal horn, corresponding to glabrous skin territory. These findings are important for interpretation of studies using molecular-genetic techniques to manipulate the functions of interneuron populations to investigate their roles in somatosensory processing.

Inhibition Mediated by Glycinergic and GABAergic Receptors on Excitatory Neurons in Mouse Superficial Dorsal Horn Is Location-Specific but Modified by Inflammation

The superficial dorsal horn is the synaptic termination site for many peripheral sensory fibers of the somatosensory system. A wide range of sensory modalities are represented by these fibers, including pain, itch, and temperature. Because the involvement of local inhibition in the dorsal horn, specifically that mediated by the inhibitory amino acids GABA and glycine, is so important in signal processing, we investigated regional inhibitory control of excitatory interneurons under control conditions and peripheral inflammation-induced mechanical allodynia. We found that excitatory interneurons and projection neurons in lamina I and IIo are dominantly inhibited by GABA while those in lamina IIi and III are dominantly inhibited by glycine. This was true of identified neuronal subpopulations: neurokinin 1 receptor-expressing (NK1R+) neurons in lamina I were GABA-dominant while protein kinase C gamma-expressing (PKCγ+) neurons at the lamina IIi-III border were glycine-dominant. We found this pattern of synaptic inhibition to be consistent with the distribution of GABAergic and glycinergic neurons identified by immunohistochemistry. Following complete Freund's adjuvant injection into mouse hindpaw, the frequency of spontaneous excitatory synaptic activity increased and inhibitory synaptic activity decreased. Surprisingly, these changes were accompanied by an increase in GABA dominance in lamina IIi. Because this shift in inhibitory dominance was not accompanied by a change in the number of inhibitory synapses or the overall postsynaptic expression of glycine receptor α1 subunits, we propose that the dominance shift is due to glycine receptor modulation and the depressed function of glycine receptors is partially compensated by GABAergic inhibition.SIGNIFICANCE STATEMENT Pain associated with inflammation is a sensation we would all like to minimize. Persistent inflammation leads to cellular and molecular changes in the spinal cord dorsal horn, including diminished inhibition, which may be responsible for enhance excitability. Investigating inhibition in the dorsal horn following peripheral inflammation is essential for development of improved ways to control the associated pain. In this study, we have elucidated regional differences in inhibition of excitatory interneurons in mouse dorsal horn. We have also discovered that the dominating inhibitory neurotransmission within specific regions of dorsal horn switches following peripheral inflammation and the accompanying hypersensitivity to thermal and mechanical stimuli. Our novel findings contribute to a more complete understanding of inflammatory pain.

Functional heterogeneity of calretinin-expressing neurons in the mouse superficial dorsal horn: implications for spinal pain processing.

The superficial spinal dorsal horn contains a heterogeneous population of neurons that process sensory inputs. Information on the properties of excitatory interneurons in this region is limited. As calretinin is a protein thought to be restricted to an excitatory population in this region, the aim of this study was to characterize calretinin-expressing neurons. Most calretinin cells (85%) exhibited large A-type potassium currents and delayed firing action potential discharge, and received strong excitatory synaptic input, whereas the remainder exhibited hyperpolarization-activated cation currents and low threshold T-type calcium currents, and tonic- or initial bursting firing patterns, and received weak excitatory synaptic input. These respective features are consistent with properties of excitatory and inhibitory interneuron populations in this region of the spinal cord. Our findings have resolved a previously unidentified population of inhibitory interneurons. Furthermore, the contrasting excitability patterns of excitatory and inhibitory calretinin-expressing neurons suggest that they play distinct roles in spinal sensory processing circuits. Neurons in the superficial dorsal horn (SDH) of the spinal cord play an important role in nociceptive, thermal, itch and light touch sensations. Excitatory interneurons comprise ∼65% of all SDH neurons but surprisingly few studies have investigated their role in spinal sensory processing. Here we use a transgenic mouse to study putative excitatory SDH neurons that express the calcium binding protein calretinin (CR). Our immunocytochemical, morphological and electrophysiological analysis identified two distinct populations of CR-expressing neurons, which we termed 'Typical' and 'Atypical'. Typical CR-expressing neurons comprised ∼85% of the population and exhibited characteristic excitatory interneuron properties including delayed firing discharge, large rapid A-type potassium currents, and central, radial or vertical cell morphologies. Atypical neurons exhibited properties consistent with inhibitory interneurons, including tonic firing or initial bursting discharge, Ih currents, and islet cell morphology. Although both Typical and Atypical CR-expressing neurons responded to noxious peripheral stimulation, the excitatory drive onto Typical CR-expressing neurons was much stronger. Furthermore, Atypical CR-expressing cells comprise at least two functionally distinct subpopulations based on their responsiveness to noxious peripheral stimulation and neurochemical profile. Together our data suggest CR expression is not restricted to excitatory neurons in the SDH. Under normal conditions, the contribution of 'Typical' excitatory CR-expressing neurons to overall SDH excitability may be limited by the presence of A-type potassium currents, which limit the effectiveness of their strong excitatory input. Their contribution may, however, be increased in pathological situations where A-type potassium currents are decreased. By contrast, 'Atypical' inhibitory neurons with their excitable phenotype but weak excitatory input may be more easily recruited during increased peripheral stimulation.

Intradermal endothelin-1 excites bombesin-responsive superficial dorsal horn neurons in the mouse.

Endothelin-1 (ET-1) has been implicated in nonhistaminergic itch. Here we used electrophysiological methods to investigate whether mouse superficial dorsal horn neurons respond to intradermal (id) injection of ET-1 and whether ET-1-sensitive neurons additionally respond to other pruritic and algesic stimuli or spinal superfusion of bombesin, a homolog of gastrin-releasing peptide (GRP) that excites spinal itch-signaling neurons. Single-unit recordings were made from lumbar dorsal horn neurons in pentobarbital-anesthetized C57BL/6 mice. We searched for units that exhibited elevated firing after id injection of ET-1 (1 μg/μl). Responsive units were further tested with mechanical stimuli, bombesin (spinal superfusion, 200 μg·ml(-1)·min(-1)), heating, cooling, and additional chemicals [histamine, chloroquine, allyl isothiocyanate (AITC), capsaicin]. Of 40 ET-1-responsive units, 48% responded to brush and pinch [wide dynamic range (WDR)] and 52% to pinch only [high threshold (HT)]. Ninety-three percent responded to noxious heat, 50% to cooling, and >70% to histamine, chloroquine, AITC, and capsaicin. Fifty-seven percent responded to bombesin, suggesting that they participate in spinal itch transmission. That most ET-1-sensitive spinal neurons also responded to pruritic and algesic stimuli is consistent with previous studies of pruritogen-responsive dorsal horn neurons. We previously hypothesized that pruritogen-sensitive neurons signal itch. The observation that ET-1 activates nociceptive neurons suggests that both itch and pain signals may be generated by ET-1 to result in simultaneous sensations of itch and pain, consistent with observations that ET-1 elicits both itch- and pain-related behaviors in animals and burning itch sensations in humans.

Properties of Sodium Currents in Neonatal and Young Adult Mouse Superficial Dorsal Horn Neurons

BackgroundSuperficial dorsal horn (SDH) neurons process nociceptive information and their excitability is partly determined by the properties of voltage-gated sodium channels. Recently, we showed the excitability and action potential properties of mouse SDH neurons change markedly during early postnatal development. Here we compare sodium currents generated in neonate (P0-5) and young adult (≥P21) SDH neurons.ResultsWhole cell recordings were obtained from lumbar SDH neurons in transverse spinal cord slices (CsF internal, 32°C). Fast activating and inactivating TTX-sensitive inward currents were evoked by depolarization from a holding potential of −100 mV. Poorly clamped currents, based on a deflection in the IV relationship at potentials between −60 and −50 mV, were not accepted for analysis. Current density and decay time increased significantly between the first and third weeks of postnatal development, whereas time to peak was similar at both ages. This was accompanied by more subtle changes in activation range and steady state inactivation. Recovery from inactivation was slower and TTX-sensitivity was reduced in young adult neurons.ConclusionsOur study suggests sodium channel expression changes markedly during early postnatal development in mouse SDH neurons. The methods employed in this study can now be applied to future investigations of spinal cord sodium channel plasticity in murine pain models.

Expression of gastrin-releasing peptide by excitatory interneurons in the mouse superficial dorsal horn.

BackgroundGastrin-releasing peptide (GRP) and its receptor have been shown to play an important role in the sensation of itch. However, although GRP immunoreactivity has been detected in the spinal dorsal horn, there is debate about whether this originates from primary afferents or local excitatory interneurons. We therefore examined the relation of GRP immunoreactivity to that seen with antibodies that label primary afferent or excitatory interneuron terminals. We tested the specificity of the GRP antibody by preincubating with peptides with which it could potentially cross-react. We also examined tissue from a mouse line in which enhanced green fluorescent protein (EGFP) is expressed under control of the GRP promoter.ResultsGRP immunoreactivity was seen in both primary afferent and non-primary glutamatergic axon terminals in the superficial dorsal horn. However, immunostaining was blocked by pre-incubation of the antibody with substance P, which is present at high levels in many nociceptive primary afferents. EGFP+ cells in the GRP-EGFP mouse did not express Pax2, and their axons contained the vesicular glutamate transporter 2 (VGLUT2), indicating that they are excitatory interneurons. In most cases, their axons were also GRP-immunoreactive. Multiple-labelling immunocytochemical studies indicated that these cells did not express either of the preprotachykinin peptides, and that they generally lacked protein kinase Cγ, which is expressed by a subset of the excitatory interneurons in this region.ConclusionsThese results show that GRP is expressed by a distinct population of excitatory interneurons in laminae I-II that are likely to be involved in the itch pathway. They also suggest that the GRP immunoreactivity seen in primary afferents in previous studies may have resulted from cross-reaction of the GRP antibody with substance P or the closely related peptide neurokinin A.

Neonatal tissue injury reduces the intrinsic excitability of adult mouse superficial dorsal horn neurons

Tissue damage during the neonatal period evokes long-lasting changes in nociceptive processing within the adult spinal cord which contribute to persistent alterations in pain sensitivity. However, it remains unclear if the observed modifications in neuronal activity within the mature superficial dorsal horn (SDH) following early injury reflect shifts in the intrinsic membrane properties of these cells. Therefore, the present study was undertaken to identify the effects of neonatal surgical injury on the intrinsic excitability of both GABAergic and presumed glutamatergic neurons within lamina II of the adult SDH using in vitro patch clamp recordings from spinal cord slices prepared from glutamic acid decarboxylase-green fluorescent protein (Gad-GFP) mice. The results demonstrate that hindpaw surgical incision at postnatal day (P) 3 altered the passive membrane properties of both Gad-GFP and adjacent, non-GFP neurons in the mature SDH, as evidenced by decreased membrane resistance and more negative resting potentials in comparison to naïve littermate controls. This was accompanied by a reduction in the prevalence of spontaneous activity within the GABAergic population. Both Gad-GFP and non-GFP neurons displayed a significant elevation in rheobase and decreased instantaneous firing frequency after incision, suggesting that early tissue damage lowers the intrinsic membrane excitability of adult SDH neurons. Isolation of inward-rectifying K+ (Kir) currents revealed that neonatal incision significantly increased Kir conductance near physiological membrane potentials in GABAergic, but not glutamatergic, lamina II neurons. Overall, these findings suggest that neonatal tissue injury causes a long-term dampening of intrinsic firing across the general population of lamina II interneurons, but the underlying ionic mechanisms may be cell-type specific.

Subpopulation‐specific patterns of intrinsic connectivity in mouse superficial dorsal horn as revealed by laser scanning photostimulation

The primary goal of this study was to map the transverse distribution of local excitatory and inhibitory synaptic inputs to mouse lamina I spinal dorsal horn neurons, using laser scanning photostimulation. A sample of lamina II neurons was also studied for comparison. Lamina I neurons received excitatory synaptic input from both laminae I-II and the outer part of III-IV, especially the II/III border region, while the inhibitory input zones were mostly confined within I-II. The excitatory synaptic input zones showed a pronounced medial asymmetry, which was correlated with a matching asymmetry in the dendritic fields of the neurons. Inhibitory input from laminae III-IV was found in a subpopulation of neurons occupying a highly restricted zone, essentially one cell layer thick, immediately below the lamina I/II border, with morphological and physiological properties that were distinct from other laminar populations in the superficial dorsal horn, and that suggest a critical role in interlaminar communication. This subpopulation also received excitatory input from laminae III-IV. Within this subpopulation, inhibitory III-IV input was correlated with the presence of long ventral dendrites. Correlations between the distribution of synaptic input zones and dendritic fields support the concept that interlaminar communication is mediated in part via contacts made onto ventrally extending dendrites of superficial laminae neurons. The results point to the presence of cell type specificity in dorsal horn circuitry, and show how the study of connectivity can itself help identify previously unrecognized neuronal populations.

Deficits in glycinergic inhibition within adult spinal nociceptive circuits after neonatal tissue damage

Tissue injury during a critical period of early postnatal development can alter pain sensitivity throughout life. However, the degree to which neonatal tissue damage exerts prolonged effects on synaptic signaling within adult spinal nociceptive circuits remains unknown. Here we provide evidence that a transient surgical injury of the hind paw during the neonatal period compromises inhibitory transmission within the adult mouse superficial dorsal horn (SDH), while the same incision occurring during the third week of life failed to evoke these long-term modifications of the SDH synaptic network. The decrease in phasic inhibitory signaling after early tissue damage reflected a selective reduction in glycine receptor (GlyR)-mediated input onto both GABAergic and presumed glutamatergic neurons within lamina II of the adult SDH. Meanwhile, neonatal incision significantly decreased the density of tonic GlyR-mediated current only in the presumed glutamatergic population during adulthood. These persistent changes in synaptic function following early injury occurred in the absence of significant alterations in the transcription of genes known to be important for glycinergic transmission. These findings suggest that aberrant sensory input during early life has permanent consequences for the functional organization of nociceptive synaptic circuits within the adult spinal cord.

Localization of neurones expressing the gap junction protein Connexin45 within the adult spinal dorsal horn: a study using Cx45-eGFP reporter mice

Connexin (Cx) proteins localized to neuronal and glial syncytia provide the ultrastructural components for intercellular communication via gap junctions. In this study, a Cx45 reporter mouse model in which the Cx45 coding sequence is substituted for enhanced green fluorescent protein (eGFP) was used to characterize Cx45 expressing neurones within adult mouse spinal cord. eGFP-immunoreactive (eGFP-IR) cells were localized at all rostro-caudal levels to laminae I–III of the dorsal horn (DH), areas associated with nociception. The neuronal rather than glial phenotype of these cells in DH was confirmed by co-localisation of eGFP-IR with the neuronal marker NeuN. Further immunohistochemical studies revealed that eGFP-IR interneurones co-express the calcium-binding protein calbindin, and to a lesser extent calretinin. In contrast, eGFP-IR profiles did not co-localize with either parvalbumin or GAD-67, both of which are linked to inhibitory interneurones. Staining with the primary afferent markers isolectin-B4 (IB4) and calcitonin gene-related peptide revealed that eGFP-IR somata within laminae I–III receive close appositions from the former, presumed non-peptidergic nociceptive afferents of peripheral origin. The presence of 5-HT terminals in close apposition to eGFP-IR interneuronal somata suggests modulation via descending pathways. These data demonstrate a highly localized expression of Cx45 in a population of interneurones within the mouse superficial dorsal horn. The implications of these data in the context of the putative role of Cx45 and gap junctions in spinal somatosensory processing and pain are discussed.

Are all spinal segments equal: intrinsic membrane properties of superficial dorsal horn neurons in the developing and mature mouse spinal cord

Neurons in the superficial dorsal horn (SDH; laminae I-II) of the spinal cord process nociceptive information from skin, muscle, joints and viscera. Most of what we know about the intrinsic properties of SDH neurons comes from studies in lumbar segments of the cord even though clinical evidence suggests nociceptive signals from viscera and head and neck tissues are processed differently. This ‘lumbar-centric' view of spinal pain processing mechanisms also applies to developing SDH neurons. Here we ask whether the intrinsic membrane properties of SDH neurons differ across spinal cord segments in both the developing and mature spinal cord. Whole cell recordings were made from SDH neurons in slices of upper cervical (C2-4), thoracic (T8-10) and lumbar (L3-5) segments in neonatal (P0-5) and adult (P24-45) mice. Neuronal input resistance (R(IN)), resting membrane potential, AP amplitude, half-width and AHP amplitude were similar across spinal cord regions in both neonates and adults (∼100 neurons for each region and age). In contrast, these intrinsic membrane properties differed dramatically between neonates and adults. Five types of AP discharge were observed during depolarizing current injection. In neonates, single spiking dominated (∼40%) and the proportions of each discharge category did not differ across spinal regions. In adults, initial bursting dominated in each spinal region, but was significantly more prevalent in rostral segments (49% of neurons in C2-4 vs. 29% in L3-5). During development the dominant AP discharge pattern changed from single spiking to initial bursting. The rapid A-type potassium current (I(Ar)) dominated in neonates and adults, but its prevalence decreased (∼80% vs. ∼50% of neurons) in all regions during development. I(Ar) steady state inactivation and activation also changed in upper cervical and lumbar regions during development. Together, our data show the intrinsic properties of SDH neurons are generally conserved in the three spinal cord regions examined in both neonate and adult mice. We propose the conserved intrinsic membrane properties of SDH neurons along the length of the spinal cord cannot explain the marked differences in pain experienced in the limbs, viscera, and head and neck.

Excitation of Mouse Superficial Dorsal Horn Neurons by Histamine and/or PAR-2 Agonist: Potential Role in Itch

Recent studies have suggested the existence of separate transduction mechanisms and sensory pathways for histamine and nonhistaminergic types of itch. We studied whether histamine and an agonist of the protease-activated receptor (PAR)-2, associated with nonhistaminergic itch, excite murine dorsal horn neurons. Single units were recorded in superficial lumbar dorsal horn of adult ICR mice anesthetized with pentobarbital. Unit activity was searched using a small intradermal hindpaw injection of histamine or the PAR-2 agonist SLIGRL-NH2. Isolated units were subsequently challenged with intradermal histamine followed by SLIGRL-NH2 (each 50 microg/1 microl) or reverse order, followed by mechanical, thermal, and algogenic stimuli. Forty-three units were classified as wide dynamic range (62%), nociceptive specific (22%), or mechano insensitive (16%). Twenty units gave prolonged (mean, 10 min) discharges to intradermal injection of histamine; 76% responded to subsequent SLIGRL-NH2, often more briefly. Units additionally responded to noxious heat (63%), cooling (43%), topical mustard oil (53%), and intradermal capsaicin (67%). Twenty-two other units gave prolonged (mean, 5 min) responses to initial intradermal injection of SLIGRL-NH2; 85% responded to subsequent intradermal histamine. They also responded to noxious heat (75%), mustard oil (93%), capsaicin (63%), and one to cooling. Most superficial dorsal horn neurons were excited by both histamine and the PAR-2 agonist, suggesting overlapping pathways for histamine- and non-histamine-mediated itch. Because the large majority of pruritogen-responsive neurons also responded to noxious stimuli, itch may be signaled at least partly by a population code.

Recording Temperature Affects the Excitability of Mouse Superficial Dorsal Horn Neurons, In Vitro

Superficial dorsal horn (SDH) neurons in laminae I-II of the spinal cord play an important role in processing noxious stimuli. These neurons represent a heterogeneous population and are divided into various categories according to their action potential (AP) discharge during depolarizing current injection. We recently developed an in vivo mouse preparation to examine functional aspects of nociceptive processing and AP discharge in SDH neurons and to extend investigation of pain mechanisms to the genetic level of analysis. Not surprisingly, some in vivo data obtained at body temperature (37 degrees C) differed from those generated at room temperature (22 degrees C) in spinal cord slices. In the current study we examine how temperature influences SDH neuron properties by making recordings at 22 and 32 degrees C in transverse spinal cord slices prepared from L3-L5 segments of adult mice (C57Bl/6). Patch-clamp recordings (KCH(3)SO(4) internal) were made from visualized SDH neurons. At elevated temperature all SDH neurons had reduced input resistance and smaller, briefer APs. Resting membrane potential and AP afterhyperpolarization amplitude were temperature sensitive only in subsets of the SDH population. Notably, elevated temperature increased the prevalence of neurons that did not discharge APs during current injection. These reluctant firing neurons expressed a rapid A-type potassium current, which is enhanced at higher temperatures and thus restrains AP discharge. When compared with previously published whole cell recordings obtained in vivo (37 degrees C) our results suggest that, on balance, in vitro data collected at elevated temperature more closely resemble data collected under in vivo conditions.

In vivo responses of mouse superficial dorsal horn neurones to both current injection and peripheral cutaneous stimulation

In the superficial dorsal horn (SDH) processing of noxious and innocuous stimuli is critically dependent on the input-output relationship of its component neurones. Such relationships are routinely examined by assessing neuronal responses to somatic current injection or activation of synaptic inputs. A more complete understanding of input-output relationships would be achieved by comparing, in the same neurone, how the two forms of activation contribute to neuronal output. Therefore, we examined how SDH neurones transform depolarizing current injections and synaptic excitation via peripheral cutaneous stimuli (brush and pinch of the hindpaw) into trains of action potentials, in an in vivo preparation of the adult mouse spinal cord. Under whole-cell current clamp recording conditions four action potential discharge patterns were observed during depolarizing current injection: tonic firing neurones (21/93) discharged spikes throughout the step; initial bursting neurones (35/93) discharged several spikes at step onset; single spiking neurones (16/93) discharged one or two spikes at step onset; and delayed firing neurones (21/93) discharged spikes delayed from the step onset. Four characteristic profiles were observed in response to application of noxious (pinch) and innocuous (brush) cutaneous stimuli: nociceptive neurones (20/37) responded maximally to pinch stimulation; light touch neurones (9/37) responded maximally to brush stimulation; subthreshold neurones (4/37) exhibited depolarizing responses without firing action potentials; and hyperpolarizing neurones (4/37) exhibited a sustained pinch-induced hyperpolarization. Comparisons of current-evoked discharge patterns with peripherally evoked responses indicate SDH neurones expressing each of the four discharge patterns could receive, and therefore participate in the processing of information concerning, either noxious or innocuous stimuli. These data suggest that a neurone's response to current injection does not necessarily help identify or predict how the same neurone will respond to physiologically or functionally relevant stimuli.

ERK integrates PKA and PKC signaling in superficial dorsal horn neurons. II. Modulation of neuronal excitability.

Protein kinases belonging to the protein kinase A (PKA), protein kinase C (PKC), and extracellular signal-related kinase (ERK) families have been identified as key players in modulating nociception at the level of the spinal cord dorsal horn, yet little is known about the effects of these kinases on membrane properties of the dorsal horn neurons. PKA, PKC, and ERK exert inhibitory effects on transient potassium currents (A-type currents or IA) in mouse superficial dorsal horn neurons (Hu et al. 2003). Here we aimed to determine the effects of these kinases on action potential firing and membrane properties of these neurons to evaluate the impact of the modulation of IA (and other conductances) in these neurons. We found that activating PKC and PKA has dramatic effects on action potential firing, reflecting an increase in the excitability of superficial dorsal horn neurons. In addition, we found that inhibitors of both PKC and ERK signaling decrease the excitability of dorsal horn neurons, suggesting that these kinases exert a tonic excitation of these cells. Consistent with our findings that these kinases inhibit A-type currents, we found that PKA, PKC, and ERK act to shorten the first-spike latency after depolarization induced by current injection. In addition, activation of these kinases increases spike frequency and action potential amplitude of dorsal horn neurons. Interestingly, we found that the effects of PKA and PKC activators are blocked by inhibitors of ERK signaling, suggesting that PKA and PKC may exert their actions by activation of ERKs.