Neurons In Intermediolateral Cell Column Research Articles

Elimination of microglia in mouse spinal cord alters the retrograde CNS plasticity observed following peripheral axon injury

Following the transection of peripherally located sympathetic preganglionic axons of the cervical sympathetic trunk (CST), transient retrograde neuronal and glial responses occur in the intermediolateral cell column (IML) of the spinal cord, the location of the parent neuronal cell bodies. The role of microglia in this central response to peripheral axon injury was examined in mice fed the PLX5622 diet containing colony-stimulating factor-1 receptor (CSF-1R) inhibitor for 28 days, which eliminated approximately 90% of spinal cord microglia. Microglia elimination did not impact baseline neurotransmitter expression in the IML neurons, and the typical neuronal plasticity observed following CST transection was unaffected. Oligodendrocyte precursor cells (OPCs) were significantly increased at one week post injury in the IML of mice fed the control diet, with no change in mature oligodendrocytes (OLs). Following microglia elimination, the baseline population of OPCs in the IML was increased, suggesting increased OPC proliferation. Injury in the microglia depleted mice resulted in no additional increase in OPCs. Though baseline astrocyte activation and GFAP protein expression were unaffected, microglia elimination led to increased activation and GFAP protein post injury when compared with mice fed the control diet. These results reveal that microglia regulate the baseline OPC population in the uninjured spinal cord and that activated microglia influence the activities of OL lineage cells as well as astrocytes. The regulatory roles of microglia observed in this study likely contribute to the long term survival of the IML neurons observed following the distal axon injury.

Retrograde influences of SCG axotomy on uninjured preganglionic neurons

There is evidence that neuronal injury can affect uninjured neurons in the same neural circuit. The overall goal of this study was to understand the effects of peripheral nerve injury on uninjured neurons located in the central nervous system (CNS). As a model, we examined whether axotomy (transection of postganglionic axons) of the superior cervical ganglion (SCG) affected the uninjured, preganglionic neurons that innervate the SCG. At 7 days post-injury a reduction in choline acetyltransferase (ChAT) and synaptophysin immunoreactivity in the SCG, both markers for preganglionic axons, was observed, and this reduction persisted at 8 and 12 weeks post-injury. No changes were observed in the number or size of the parent cell bodies in the intermediolateral cell column (IML) of the spinal cord, yet synaptic input to the IML neurons was decreased at both 8 and 12 weeks post-injury. In order to understand the mechanisms underlying these changes, protein levels of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) were examined and reductions were observed at 7 days post-injury in both the SCG and spinal cord. Taken together these results suggest that axotomy of the SCG led to reduced BDNF in the SCG and spinal cord, which in turn influenced ChAT and synaptophysin expression in the SCG and also contributed to the altered synaptic input to the IML neurons. More generally these findings provide evidence that the effects of peripheral injury can cascade into the CNS and affect uninjured neurons.

Transection of preganglionic axons leads to CNS neuronal plasticity followed by survival and target reinnervation

The goals of the present study were to investigate the changes in sympathetic preganglionic neurons following transection of distal axons in the cervical sympathetic trunk (CST) that innervate the superior cervical ganglion (SCG) and to assess changes in the protein expression of brain derived neurotrophic factor (BDNF) and its receptor TrkB in the thoracic spinal cord. At 1 week, a significant decrease in soma volume and reduced soma expression of choline acetyltransferase (ChAT) in the intermediolateral cell column (IML) of T1 spinal cord were observed, with both ChAT-ir and non-immunoreactive neurons expressing the injury marker activating transcription factor 3. These changes were transient, and at later time points, ChAT expression and soma volume returned to control values and the number of ATF3 neurons declined. No evidence for cell loss or neuronal apoptosis was detected at any time point. Protein levels of BDNF and/or full length TrkB in the spinal cord were increased throughout the survival period. In the SCG, both ChAT-ir axons and ChAT protein remained decreased at 16 weeks, but were increased compared to the 10 week time point. These results suggest that though IML neurons show reduced ChAT expression and cell volume at 1 week following CST transection, at later time points, the neurons recovered and exhibited no significant signs of neurodegeneration. The alterations in BDNF and/or TrkB may have contributed to the survival of the IML neurons and the recovery of ChAT expression, as well as to the reinnervation of the SCG.

Angiotensin type 2 receptors in the intermediolateral cell column of the spinal cord: Negative regulation of sympathetic nerve activity and blood pressure

Our previous study demonstrated that AT2R in brainstem nuclei participated in the regulation of sympathetic outflow and cardiovascular function. However, the functional significance of AT2R in the intermediolateral cell column (IML) of the thoracic spinal cord in normal rats remains elusive. We hypothesized that AT2R activation in the IML exerts a sympatho-inhibitory effect. Using Western-blot analysis, immunohistochemical staining and quantitative real-time PCR, both AT1R and AT2R expressions were detected in the spinal cord. The highest AT2R protein expression was found in the IML, while AT1R expression didn't display regional differences within the gray matter. Microinjection of Ang II into the IML dose-dependently elevated mean blood pressure (MAP, employing a transducer-tipped catheter) and renal sympathetic nerve activity (RSNA, using a pair of platinum-iridium recording electrodes), which were completely abolished by Losartan, and attenuated by TEMPOL and apocynin. Activation of AT2R in the IML with CGP42112 evoked hypotension (ΔMAP: -21 ± 4 mmHg) and sympatho-inhibition (RSNA: 73 ± 3% of baseline), which were completely abolished by PD123319 and l-NAME. Blockade of AT2R in the IML with PD123319 significantly increased MAP (11 ± 1 mmHg) and sympathetic nerve activity (RSNA: 133 ± 13% of baseline). Moreover, PD123319 significantly enhanced the Ang II induced pressor response. Furthermore, in isolated IML neurons, CGP42112 treatment augmented potassium current and decreased resting membrane potential by employing whole-cell patch clamp. In the normal condition, AT2R in the IML tonically inhibits sympathetic activity through an NO/NOS dependent pathway and subsequent potassium channel activation.

Effects of corticotropin-releasing factor on intermediolateral cell column neurons of newborn rats

Corticotropin-releasing factor (CRF) is a neuropeptide that mediates neuroendocrine, autonomic, and behavioral processes associated with the stress response. CRF-containing fibers and receptors are found in various regions of the central nervous system including the spinal cord. Here, we report excitatory effects of CRF on sympathetic preganglionic neurons in the intermediolateral cell column (IML) of in vitro spinal cord preparations from newborn rats. We also examine the receptor subtypes that are involved in the CRF effects. Application of CRF significantly depolarized the IML neurons and increased the frequency of excitatory postsynaptic potentials (EPSPs) in the IML neurons. These effects were blocked by the CRF receptor 1 antagonist, antalarmin. Menthol, a transient receptor potential channel M8 agonist, depressed EPSPs enhanced by CRF. Our findings suggested that CRF depolarized the IML neurons via direct postsynaptic action and CRF-affected interneurons located in the spinal cord send EPSPs to IML neurons. These excitatory effects of CRF may be caused through CRF1 receptors but not CRF2 receptors.

284 EFFECTS OF CORTICOTROPIN-RELEASING FACTOR ON INTERMEDIOLATERAL CELL COLUMN NEURONS

Background: CRF receptors are widely distributed in the central nervous system including the spinal cord. Although CRF indirectly participates in control of the blood pressure, it is unknown whether CRF is involved in blood pressure control via direct action on central neurons. Here, we report effects of CRF on sympathetic preganglionic neurons in the intermediolateral cell column (IML) of the spinal cord. Methods: Transverse slices of Th2 segment from newborn rats (0-4 days old) were superfused by artificial cerebrospinal fluid. Membrane potentials of IML neurons were recorded by whole-cell patch-clamp. Preganglionic neurons were confirmed by antidromic action potential induced by Th2 ventral root stimulation and cell staining by Lucifer Yellow. CRF (50 nM) was bath-applied. We measured the membrane potential change and counted the number of excitatory postsynaptic potentials (EPSPs) of the IML neurons. Results: The frequency of EPSPs in the IML neurons was 3.67 ± 3.57 Hz (n = 9) in control solution. Application of CRF increased the number of EPSPs (220%) and induced membrane depolarization by 2.4 ± 1.3 mV. The increase of EPSPs number by CRF was attenuated by pretreatment of a CRF-1 antagonist, antalarmin (124%) but not by pretreatment of a CRF-2 antagonist, astressin2B (268). Conclusion: Our findings suggest that CRF affected directly IML neurons and interneurons in the spinal cord. Moreover, CRF exerted the excitatory effect on the IML neurons through CRF-1 receptors, whereas effect via CRF-2 might be rather inhibitory. Thus, CRF-related drugs are expected as one of the choices of the pharmacologic control of the blood pressure.

Imbalance of Angiotensin Receptor Expression and Function in the Spinal Cord: Potential Mechanism of Sympathetic Overactivity in CHF Rats

In a recent study we showed that angiotensin II (AII) in the intermediolateral cell column (IML) of the spinal cord plays a critical role in the regulation of mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) in normal rats. In this study, the role of IML AII in sympatho‐excitation of chronic heart failure (CHF) rat induced by coronary‐ligation was studied. AT1R and AT2R expression in the IML was measured by Western blot and immunostaining. Compared with sham rats, CHF rats exhibited a higher AT1R and lower AT2R expression. Then effects of microinjecting AII, losartan (AT1R blocker), or PD123319 (AT2R blocker) into the IML were evaluated. AII evoked significantly stronger pressor and sympatho‐excitatory responses in CHF rats than that in sham rats (ΔMAP: 26 ± 1 vs 15 ± 2 mmHg, RSNA: 210 ± 9 vs 142 ± 6 % of baseline). Losartan significantly decreased basal RSNA and attenuated AII evoked responses in CHF rats, while PD123319 failed to affect MAP and RSNA. Finally, the effect of AII on electrophysiological properties of cultured IML neurons was assessed using whole‐cell patch clamp. AII significantly inhibited the K+ current (from 167 ± 9 to 39 ± 5 pA/pF at +80 mV) and elevated the resting membrane potential (from −55 ± 1 to −31 ± 1 mV). In conclusion, the change of AT1R and AT2R expression in the IML contributes to the sympatho‐excitation in CHF, possibly by elevating neuronal excitability via K+ current inhibition.

Electrophysiological characteristics of identified kidney-related neurons in adult rat spinal cord slices

Whole-cell patch-clamp recordings were made from kidney-related neurons in the intermediolateral cell column (IML) in horizontal slices of thoracolumbar spinal cord from adult rats. Kidney-related neurons were identified in vitro subsequent to inoculation of the kidney with a fluorescent, retrograde, transynaptic pseudorabies viral label (i.e., PRV-152). Kidney-related neurons detected in the IML expressed choline acetyltransferase, characteristic of spinal preganglionic motor neurons. Their mean resting potential was −51±4mV and input resistance was 448±39MΩ. Both spontaneous inhibitory and excitatory post-synaptic currents (i.e., sIPSCs and sEPSCs) were observed in all neurons. The mean frequency for sEPSCs (3.1±1Hz) was approximately 2.5 times that for sIPSCs (1.4±0.3Hz). Application of the glycine and GABAA receptor-linked Cl− channel blocker, picrotoxin (100μM) blocked sIPSCs, while the ionotropic glutamate receptor antagonist, kynurenic acid (1mM) blocked all sEPSCs, indicating they were mediated by GABA/glycine and glutamate receptors, respectively. Thus, using PRV-152 labeling allowed whole-cell patch-clamp recording of neurons in the adult spinal cord, which were kidney-related. Excitatory glutamatergic input dominated synaptic responses in these cells, the membrane characteristics of which resembled those of immature IML neurons. Combined PRV-152 pre-labeling and whole-cell patch-clamp recordings may allow more effective analysis of synaptic plasticity seen in adult models of injury or chronic disease.

Involvement of protein kinase C and Src tyrosine kinase in acute tolerance to ethanol inhibition of spinal NMDA‐induced pressor responses in rats

The present study was carried out to examine the role of protein kinases in the development of acute tolerance to the effects of ethanol on spinal N-methyl-D-aspartate (NMDA) receptor-mediated pressor responses during prolonged ethanol exposure. Blood pressure responses induced by intrathecal injection of NMDA were recorded. The levels of several phosphorylated residues on NMDA receptor NR1 (GluN1) (NR1) and NMDA receptor NR2B (GluN2B) (NR2B) subunits were determined by immunohistochemistry and Western blot analysis. Ethanol inhibited spinal NMDA-induced pressor responses at 10 min, but the inhibition was significantly reduced at 40 min following continuous infusion. This effect was dose-dependently blocked by chelerythrine [a protein kinase C (PKC) inhibitor, 1-1000 pmol] or PP2 (a Src family tyrosine kinase inhibitor, 1-100 pmol) administered intrathecally 10 min following ethanol infusion. A significant increase in the immunoreactivity of phosphoserine 896 of NR1 subunits (pNR1-Ser896) and phosphotyrosine 1336 of NR2B subunits (pNR2B-Tyr1336) was found in neurons of intermediolateral cell column during the development of tolerance. Levels of pNR1-Ser896 and pNR2B-Tyr1336 were also significantly increased in lateral horn regions of the spinal cord slices incubated with ethanol for 40 min in vitro. The increases in pNR1-Ser896 and pNR2B-Tyr1336 levels were inhibited by post-treatment with chelerythrine and PP2, respectively, both in the in vivo and in vitro studies. The results suggest that activation of PKC and Src tyrosine kinase during prolonged ethanol exposure leading to increases in the levels of pNR1-Ser896 and pNR2B-Tyr1336 may contribute to acute tolerance to inhibition by ethanol of NMDA receptor function.

Electrophysiological responses of sympathetic preganglionic neurons to ANG II and aldosterone

The intermediolateral cell column (IML) of the spinal cord is an important area where sympathetic impulses propagate to peripheral sympathetic organs. ANG II and aldosterone are important components of the renin-angiotensin-aldosterone system (RAAS), which activate the sympathetic nervous system. Each is partly synthesized in the brain and plays a paracrine role in the regulation of blood pressure independently of RAAS in the periphery. Our purpose in the present study was to clarify the contributions of sympathetic preganglionic neurons in the IML (IML neurons) and the effects of ANG II and aldosterone on the sympathetic nervous system. To examine responses to ANG II and aldosterone, we intracellularly recorded 104 IML neurons using a whole cell patch-clamp technique in spinal cord slice preparations. IML neurons were classified into two types: silent and firing. Both neuron types were significantly depolarized by ANG II, and candesartan inhibited this depolarization. After pretreatment with TTX, firing neurons (but not silent neurons) were significantly depolarized by ANG II. Aldosterone significantly increased the number of excitatory postsynaptic potentials (EPSPs) in both neuron types, but this response disappeared after pretreatment with TTX. ANG II and aldosterone had no synergistic effects on the IML neurons. The silent neurons had large cell soma, and many more dendrites than the firing neurons. These results suggest that ANG II acts presynaptically and postsynaptically in IML neurons, while aldosterone acts mainly presynaptically. Thus, the physiological effects of these substances are likely to be transmitted via specific membrane receptors of IML and/or presynaptic neurons.

Urocortin 3 Microinjected Into The Intermediolateral Cell Column Elicits Tachycardia In The Rat

Urocortin 3 (Ucn 3) is a new member of corticotropin releasing factor (CRF) family of peptides. There is limited information about the cardiovascular actions of this peptide. Urethane‐anesthetized, artificially ventilated, adult male Wistar rats, weighing between 325‐350 gm, were used in this study. L‐Glutamate (L‐Glu; 5 mM), microinjected (30 nl) into the right intermediolateral cell column (IML) at T1 or T2 level, elicited an increase (35 ± 4 bpm) in heart rate (HR). The onset and duration of these responses was 5 and 50‐60 sec, respectively. Microinjections (30 nl) of Ucn 3 (0.31, 0.62, 0.12 and 0.25 mM) into the same sites in the IML elicited tachycardia; the increases in HR were 13.6 ± 1.5, 31.8 ± 3.9, 37.9 ± 2.1 bpm and 33.6 ± 3.4 bpm, respectively. Maximum tachycardic responses were elicited by 0.12 mM concentration of Ucn 3. The tachycardic responses elicited by Ucn 3 (0.12 mM) were significantly attenuated by prior microinjections (30 nl) of a selective antagonist (K41498, 1 mM) for CRF type 2 receptors. It was concluded that Ucn 3 exerts an excitatory effect on IML neurons controlling cardiac function.Support: NIH grants HL024347 and HL076248.

Characterization of trkB immunoreactive cells in the intermediolateral cell column of the rat spinal cord.

The objective of the present study was to characterize the trkB receptor immunoreactive (-ir) cells in the intermediolateral cell column (IML) of the upper thoracic spinal cord. Small trkB-ir cells (area=56.1+/-4.4 microm(2)) observed in the IML showed characteristics of oligodendrocytes and were frequently observed in close apposition to choline acetyltransferase (ChAT)-ir cell bodies. Large trkB-ir cells (area=209.3+/-25.2 microm(2)) showed immunoreactivity for the neuronal marker NeuN, indicating their neuronal phenotype, as well as for ChAT, a marker for preganglionic neurons. TrkB and ChAT were co-localized in IML neurons primarily in cases that had received in vivo administration of nerve growth factor (NGF). These findings reveal two different cell types, oligodendrocytes and neurons, in the IML of the spinal cord that show trkB immunoreactivity, suggesting their regulation by brain derived neurotrophic factor (BDNF) and/or neurotrophin-4 (NT-4). In addition, there is evidence that NGF may play a role in the regulation of trkB-ir preganglionic neurons in the IML.

Novel axonal projection from the caudal end of the ventrolateral medulla to the intermediolateral cell column

We used an optical imaging technique to investigate whether axons of neurons in the caudal end of the ventrolateral medulla (CeVLM), as well as axons of neurons in the rostral ventrolateral medulla (RVLM), project to neurons in the intermediolateral cell column (IML) of the spinal cord. Brain stem-spinal cord preparations from neonatal normotensive Wistar-Kyoto and spontaneously hypertensive rats were stained with a voltage-sensitive dye, and responses to electrical stimulation of the IML at the Th2 level were detected as changes in fluorescence intensity with an optical imaging apparatus (MiCAM-01). The results were as follows: 1) depolarizing responses to IML stimulation during low-Ca high-Mg superfusion were detected on the ventral surface of the medulla at the level of the CeVLM, as well as at the level of the RVLM, 2) depolarizing responses were also detected on cross sections at the level of the CeVLM, and they had a latency of 24.0 +/- 5.5 (SD) ms, 3) antidromic action potentials in response to IML stimulation were demonstrated in the CeVLM neurons where optical images were detected, and 4) glutamate application to the CeVLM increased the frequency of excitatory postsynaptic potentials (EPSPs) and induced depolarization of the IML neurons. The optical imaging findings suggested a novel axonal and functional projection from neurons in the CeVLM to the IML. The increase in EPSPs of the IML neurons in response to glutamate application suggests that the CeVLM participates in the regulation of sympathetic nerve activity and blood pressure and may correspond to the caudal pressor area.

DIFFERENTIAL CONTRIBUTION OF N-METHYL-d-ASPARTATE AND NON-N-METHYL-d-ASPARTATE RECEPTORS IN THE INTERMEDIOLATERAL CELL COLUMN OF THE THORACIC SPINAL CORD TO SYMPATHETIC VASOMOTOR TONE DURING EXPERIMENTAL ENDOTOXEMIA IN THE RAT

Although the rostral ventrolateral medulla maintains neurogenic vasomotor tone via glutamatergic excitation to sympathetic preganglionic neurons located at the intermediolateral cell column (IML) of thoracic spinal cord, the relative contribution of N-methyl-d-aspartate (NMDA) and non-NMDA receptors on IML neurons at rest or during endotoxemia remain unknown. The present study addressed this issue using a combination of physiological, pharmacological, and double-immunofluorescence approaches. Adult male Sprague-Dawley rats maintained under propofol anesthesia were used. Intrathecal administration of equimolar concentrations (75, 150, or 300 nmol) of an NMDA antagonist, dizocilpine (MK801), or a non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, into T10 to T12 spinal cord elicited a reduction in resting vasomotor tone that was comparable in time course and in magnitude. Although both glutamate receptor antagonists exacerbated mortality and potentiated the elicited hypotension, bradycardia, or reduction in vasomotor tone during experimental endotoxemia induced by intravenous administration of Escherichia coli lipopolysaccharide (30 mg kg-1; results comparable to 6-cyano-7-nitroquinoxaline-2,3-dione at 150 nmol) were obtained only when MK801 was given at 300 nmol. Confocal microscopy further showed that augmented immunoreactivity of NR1 subunit on IML neurons coincided with the phase of endotoxemia when vasomotor tone was augmented; GluR1 immunoreactivity remained stable throughout experimental endotoxemia. These findings suggest that NMDA and non-NMDA receptors on IML neurons contribute to the generation of resting sympathetic vasomotor tone. However, up-regulation of NMDA receptors on IML neurons plays a crucial role in the maintenance of vasomotor tone during endotoxemia.

A monosynaptic connection between baroinhibited neurons in the RVLM and IML in Sprague-Dawley rats

To date, few studies have examined the relationship between the firing rate of neurons in the rostral ventrolateral medulla (RVLM) and neurons in the intermediolateral cell column (IML) of the spinal cord. In 19 Sprague-Dawley rats, the relationship between 20 pairs of baroinhibited RVLM and IML units was analyzed by cross-correlation. Three criteria were applied before acceptance that the firing rate of a pair of neurons was correlated. First, at an appropriate latency following the firing of an RVLM neuron, as judged from previous studies (4–200 ms), the peak in the firing rate of an IML neuron was approximately double that of the averaged surrounding bin counts. Secondly, the peak grew steadily in the examined period. Thirdly, the peak was restricted to a 1-ms bin. With this approach, a correlation was found between RVLM and IML neurons in 3 pairs in all. In 2 pairs, a correlation was found at basal arterial pressure (AP). When AP was decreased using a caval snare, a correlation was demonstrated in a further pair. A possible potentiation of synaptic strength during hypotensive stimuli is discussed.

Pituitary adenylate cyclase activating polypeptide (PACAP) expression in sympathetic preganglionic projection neurons to the superior cervical ganglion

Pituitary adenylate cyclase activating polypeptides (PACAP27 and PACAP38) are members of the VIP/secretin/glucagon family of peptides and have diverse neuroregulatory effects in sympathoadrenal cell development and function. PACAP peptides regulate rat superior cervical ganglion (SCG) neuron catecholamine and neuropeptide Y content and secretion, and promote sympathoneuroblast survival through activation of specific PACAP1 receptor isoforms. In examining the potential sources of PACAP regulating the SCG, PACAP expression was identified in rat preganglionic neurons in the intermediolateral cell column (IML) of the thoracic spinal cord which provide primary afferent projections to this sympathetic ganglion. Thoracic spinal cord segments (T1-4) contained approximately 17 pmol PACAP38 immunoreactivity/g tissue wet weight. Reverse-transcription polymerase chain reaction of cDNA from microdissected thoracic spinal cord using primers specific for rat neuronal proPACAP identified proPACAP mRNA expression in the IML; the results correlated with neurons labeled for proPACAP mRNA by in situ hybridization histochemistry and implicated PACAP biosynthesis in IML neurons. To demonstrate directly proPACAP transcript expression in preganglionic projection neurons to the SCG, the ganglion was decentralized and the sympathetic trunk immersed in fluorogold to identify sympathetic preganglionic neurons by retrograde labeling. Cryosections of spinal cord segments containing preganglionic neuron fluorogold labeled neurons were processed subsequently for in situ hybridization histochemical localization of proPACAP mRNA using a digoxigenin-labeled riboprobe; IML neurons were examined for fluorogold and digoxigenin/alkaline phosphatase product dual labeling. More than half of the preganglionic projection neurons to the SCG expressed PACAP mRNA, consistent with the postulate that PACAP peptides released from a subpopulation of thoracic IML preganglionic neurons may be physiological anterograde modulators of sympathetic SCG function.

Pituitary adenylate cyclase activating polypeptide (PACAP) expression in sympathetic preganglionic projection neurons to the superior cervical ganglion

Pituitary adenylate cyclase activating polypeptides (PACAP27 and PACAP38) are members of the VIP/secretin/glucagon family of peptides and have diverse neuroregulatory effects in sympathoadrenal cell development and function. PACAP peptides regulate rat superior cervical ganglion (SCG) neuron catecholamine and neuropeptide Y content and secretion, and promote sympathoneuroblast survival through activation of specific PACAP1 receptor isoforms. In examining the potential sources of PACAP regulating the SCG, PACAP expression was identified in rat preganglionic neurons in the intermediolateral cell column (IML) of the thoracic spinal cord which provide primary afferent projections to this sympathetic ganglion. Thoracic spinal cord segments (T1–4) contained approximately 17 pmol PACAP38 immunoreactivity/g tissue wet weight. Reverse-transcription polymerase chain reaction of cDNA from microdissected thoracic spinal cord using primers specific for rat neuronal proPACAP identified proPACAP mRNA expression in the IML; the results correlated with neurons labeled for proPACAP mRNA by in situ hybridization histochemistry and implicated PACAP biosynthesis in IML neurons. To demonstrate directly proPACAP transcript expression in preganglionic projection neurons to the SCG, the ganglion was decentralized and the sympathetic trunk immersed in fluorogold to identify sympathetic preganglionic neurons by retrograde labeling. Cryosections of spinal cord segments containing preganglionic neuron fluorogold labeled neurons were processed subsequently for in situ hybridization histochemical localization of proPACAP mRNA using a digoxigenin-labeled riboprobe; IML neurons were examined for fluorogold and digoxigenin/alkaline phosphatase product dual labeling. More than half of the preganglionic projection neurons to the SCG expressed PACAP mRNA, consistent with the postulate that PACAP peptides released from a subpopulation of thoracic IML preganglionic neurons may be physiological anterograde modulators of sympathetic SCG function. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 325–336, 1998

The intermediolateral cell column of the thoracic spinal cord is comprised of target-specific subnuclei: evidence from retrograde transport studies and immunohistochemistry

In this study we examined the hypothesis that the intermediolateral cell column (IML) of the thoracic spinal cord, the nucleus from which preganglionic sympathetic neurons originate, provides an anatomical substrate through which selective regulation of sympathetic nervous system targets is accomplished. Preganglionic sympathetic neurons of rats were retrogradely labeled by the simultaneous exposure of the cervical sympathetic trunk (CST) and the adrenal medulla to Fluoro-Gold and True blue, contrasting fluorescent dyes. Retrograde labeling from these sites revealed 2 populations of sympathetic preganglionic neurons in IML whose distribution overlapped between segments T1 and T4. In regions where these 2 groups of retrogradely labeled neurons overlapped, sympathoadrenal preganglionic (SAP) neurons occupied the most lateral aspect of the nucleus. It was also determined whether individual retrogradely labeled neurons within these two groups sent axon collaterals to both the CST and adrenal medulla. Diamidino yellow, a fluorescent retrograde tracer dye that labels only nuclei, was substituted for Fluoro-Gold and used in combination with True blue to simultaneously label preganglionic sympathetic neurons projecting to either the CST or adrenal medulla. No double-labeled cell bodies were observed in spinal cords of rats treated in this manner. Thus it appeared that the efferent projections of these 2 cell populations in IML were target-specific. Immunohistochemical analysis of the relationship between nerve fibers in the IML and preganglionic sympathetic neurons was also undertaken in an attempt to classify further these 2 populations of sympathetic preganglionic neurons. Equal proportions of identified CST and SAP neurons appeared to be apposed by varicosities immunoreactive for either somatostatin or serotonin. On the other hand, when the comparison was based on whether oxytocin-immunoreactive varicosities appeared to appose these 2 populations of retrogradely labeled sympathetic neurons, a highly significant difference was revealed. That is, oxytocin-immunoreactive fibers and terminals appeared to avoid SAP neurons. Thus these data support the hypothesis that an anatomical substrate exists in spinal cord IML whereby selective regulation of sympathetic nervous system targets may be mediated. Moreover, the lack of oxytocin-immunoreactive varicosities apposing SAP neurons in IML suggests that if the paraventricular nucleus innervates SAP neurons in IML, it does so via a population of neurons that do not use oxytocin as a neurotransmitter.

Thyrotropin-releasing hormone in spinal cord: coexistence with serotonin and with substance P in fibers and terminals apposing identified preganglionic sympathetic neurons

In this study we utilized the technique of simultaneous immunofluorescent double-labeling to investigate possible coexistence of the putative neurotransmitter thyrotropin-releasing hormone (TRH) with serotonin (5-HT) and with substance P (SP) in the intermediolateral cell column (IML) of rat spinal cord. We observed fibers and terminals immunoreactive for both TRH and 5-HT or TRH and SP in IML. In addition, this technique was used in animals in which we retrogradely labeled, with fluorescent tracer dyes, preganglionic sympathetic neurons within IML from either the adrenal medulla or the proximal cut end of the cervical sympathetic trunk. In these animals, fibers and terminals containing these combinations of neurotransmitters appeared to appose identified preganglionic sympathetic neurons in IML. These data represent the first direct immunohistochemical demonstration of fibers and terminals in spinal cord which display coexistence of TRH- with either 5-HT- or SP-immunoreactivity. In addition, the proximity of TRH-immunoreactive fibers and terminals to sympathetic preganglionic neurons in IML support a role for TRH in the regulation of central sympathetic outflow.

Coexistence of serotonin- and substance P-like immunoreactivity in nerve fibers apposing identified sympathoadrenal preganglionic neurons in rat intermediolateral cell column

In this study we examined the possibility that serotonin (5-HT) and substance P (SP) coexist in fibers and terminals afferent to sympathoadrenal preganglionic (SAP) neurons in the intermediolateral cell column (IML) of the spinal cord. SAP neurons in the IML were identified by retrograde labeling with either Fast Blue or True Blue injected into the adrenal medulla of rats. A simultaneous immunofluorescent double labeling technique was used to identify both 5-HT- and SP-like immunoreactivity in single tissue sections. Labeled SAP neurons were observed which were apposed by fibers immunoreactive for either neurotransmitter, as well as SAP neurons apposed by neither 5-HT- nor SP-like immunoreactive structures. In addition, 5-HT- and SP-like immunoreactivity were observed in separate fibers apposing the same labeled neuron and coexisting in fibers and terminal appearing in apposition to labeled SAP neurons. These data suggest a complex interaction by these neurotransmitters in regulating sympathetic outflow and may provide a model for interpreting conflicting observations concerning the effects of local 5-HT administration on sympathetic nerve activity.