Isolectin B4 (IB4)-conjugated streptavidin for the selective knockdown of proteins in IB4-positive (+) nociceptors.

In vivo analysis of protein function in nociceptor subpopulations using antisense oligonucleotides and short interfering RNAs is limited by their non-selective cellular uptake. To address the need for selective transfection methods, we covalently linked isolectin B4 (IB4) to streptavidin and analyzed whether it could be used to study protein function in IB4(+)-nociceptors. Rats treated intrathecally with IB4-conjugated streptavidin complexed with biotinylated antisense oligonucleotides for protein kinase C epsilon (PKCε) mRNA were found to have: a) less PKCε in dorsal root ganglia (DRG), b) reduced PKCε expression in IB4(+) but not IB4(-) DRG neurons, and c) fewer transcripts of the PKCε gene in the DRG. This knockdown in PKCε expression in IB4(+) DRG neurons is sufficient to reverse hyperalgesic priming, a rodent model of chronic pain that is dependent on PKCε in IB4(+)-nociceptors. These results establish that IB4-streptavidin can be used to study protein function in a defined subpopulation of nociceptive C-fiber afferents.

Cortical modulation of pain.

The sensation commonly referred to as 'pain' has two components. The first is the sensory-discriminative component and provides information on location, modality and intensity of stimuli. The second is the affective-motivational component and refers to the emotional responses (fear, distress etc.) and the urge to respond evoked by the somatic sensation, and at the cortical level these two components appear to be located in different regions. The cortex probably influences pain by two different mechanisms. There is good evidence that the cortex can reduce pain by interrupting the transmission of noxious information from the spinal cord level by activating descending pain modulatory systems located in the brainstem. Less well established is the idea that modulation can also occur at the cortical level to change the affective-motivational aspects of nociception so that pain is perceived but looses its emotional and aversive component.

GABA puts a stop to pain.

A lack of inhibition, particularly that mediated by gamma-amino butyric acid (GABA), the main inhibitory transmitter of the central nervous system (CNS), is responsible for many pain states. Until recently, few GABA acting drugs were available and were prescribed mostly for relieving muscle spasms, anxiety and epilepsy, but rarely for pain. The basic metabolic pathway of GABA is well known and we are now beginning to understand the function of this neurotransmitter in the complex circuitry underlying pain, especially in the context of nerve injury. Analgesic compounds are now being developed targeting GABA transporters as well as GABA associated enzymes and receptors. Some GABA analogs act by inhibiting ion channels, a property that contributes to their analgesic effects. However, despite considerable progress in developing new compounds, the use of systemically acting GABAergic drugs is limited by unwanted side-effects on systems other than those involved in pain, and by the fact that in certain areas of the brain, GABA can enhance rather than reduce pain. The advent of new drugs targeting subtypes of GABA receptors and transporters and the possibility of using newly developed delivery systems, such as intrathecal pumps and viral vectors, to target specific areas of the nervous system will likely help circumvent these problems.

Long-term intrathecal catheterization in the rat.

We report an intrathecal (i.t.) catheter system that permits repeated administration of volumes of 10 microl or more in the awake rat over many months. A small skin incision is made and a 32 ga polyurethane catheter is inserted in the sacral subarachnoid space using a modified 22 ga needle. The other end of the catheter is tunneled subcutaneously to the flank and exteriorized through a titanium port. The device is well tolerated, does not cause sensory or motor deficits, and does not interfere with behavioral testing. Rats equipped with this device can be housed with other rats. Over the 9 month observation period the function of the catheter was verified by repeated injection of 15 microl of 2% lidocaine that caused temporary paraplegia. Out of 12 implanted rats, the number of fully functional catheters was 10 at 3 months, seven at 6 months, and six at 9 months. At 3 months, i.t. injection of anti-dopamine-beta-hydroxylase antibodies conjugated to saporin (DBH-SAP, 5 microg/10 microl) resulted in noradrenergic denervation of the spinal cord in all rats (n=10). We propose that intrathecal catheterization is well suited for long term behavioral and pharmacological studies.

Quantitative analysis of the dendrites of sacral preganglionic neurons in the cat.

Quantitative analyses were performed on the dendrites and somata of 25 electrophysiologically identified preganglionic neurons (PGN) obtained from the sacral spinal cord of the cat by intracellular injection of Neurobiotin or horseradish peroxidase. Total dendritic length and surface area were measured for each dendrite. The sizes of the stem dendrites measured at their base were positively correlated with the sizes of the entire tree and numbers of end branches. Total surface area of somata and dendrites averaged 39,138 microm(2); 90.7% of that was from the dendrites. To obtain measurements of the relative contributions of PGN dendrites to specific regions of the spinal cord, the percentage of each dendrite occupying eight spinal cord regions was recorded. Sixty-three percent of the dendrites projected dorsal to their somata, whereas an average of 33.3% of dendrites were located in the white matter, most of them in the lateral and dorsolateral funiculi. The neurons within this sample formed a continuum with some neurons having a large percentage of dendrites in lamina I but little in the white matter, whereas at the other end of the continuum were cells with the reverse configuration. The intermediate neurons had dendrites in both locations. Taken together, these data indicate a heterogeneous population of PGN in the lateral band of the sacral parasympathetic nucleus.

Two paths for dissemination of Herpes simplex virus from infected trigeminal ganglion to the murine cornea.

Herpes simplex virus type 1 (HSV) was introduced into the mouse trigeminal ganglion by stereotaxic injection. We examined the form in which the virus was transported anterograde within axons and the spread of virus to glial and endoneurial cells of the nerve using EM immunocytochemistry. Our results indicate that viral dissemination in the trigeminal nerve may occur both within the axon and in the extracellular space of the endoneurium. HSV is intraaxonally transported at least in part as a nucleocapsid, i.e., with neither viral envelope nor additional cellular membranes. Schwann cells are infected as a result of spread in the endoneurium, as well as by nearby axons.

CNS induced neurogenic cystitis is associated with bladder mast cell degranulation in the rat.

PURPOSE: To determine if bladder mast cell degranulation is involved in the genesis of neurogenic cystitis induced by pseudorabies virus (PRV) invasion of the central nervous system (CNS). MATERIALS AND METHODS: Rats received a total of 4 x 106 plaque forming units (pfu) of PRV-Bartha in the abductor caudalis dorsalis (ACD) muscle. Granulated bladder mast cells per mm2 of bladder tissue and urine histamine content were monitored as the cystitis developed over the next few days. In a subgroup of rats, intravesical resiniferatoxin was used to remove capsaicin-sensitive sensory bladder afferents, while another subgroup was pretreated with a mast cell degranulator. RESULTS: PRV injection into the ACD muscle leads to neurogenic cystitis. Histamine levels were elevated in the urine of virus injected rats before any behavioral or microscopical signs of cystitis were present. When the cystitis became clinically manifest, urine histamine returned to control levels, and the number of granulated mast cells dropped significantly. Rats in which capsaicin-sensitive afferents had been removed did not show any signs of cystitis, or increase in urine histamine, or change in the number of granulated mast cells. Pretreatment of animals with a mast cell degranulator completely prevented the appearance of cystitis without altering the CNS disease. CONCLUSION: These results provide further evidence that mast cells are involved in neurogenic cystitis induced by changes in CNS activity.

The spread of herpes simplex virus type 1 from trigeminal neurons to the murine cornea: an immunoelectron microscopy study.

An animal model has been developed to clarify the mechanism for spread of herpes simplex virus (HSV) from neuron to epithelial cells in herpetic epithelial keratitis. HSV was introduced into the murine trigeminal ganglion via stereotaxic guided injection. After 2 to 5 days, the animals were euthanized. Ganglia and corneas were prepared for light and electron microscopic immunocytochemistry with antisera to HSV. At 2 days, labeled axons were identified in the stromal layer. At 3 days, we could detect immunoreactive profiles of trigeminal ganglion cell axons that contained many vesicular structures. By 3 and 4 days, the infection had spread to all layers of epithelium, and the center of a region of infected epithelium appeared thinned. At 5 day, fewer basal cells appeared infected, although infection persisted in superficial cells where it had expanded laterally. Mature HSV was found in the extracellular space surrounding wing and squamous cells. Viral antigen was expressed in small pits along the apical surfaces of wing and squamous cells but not at the basal surface of these cells or on basal cells. This polarized expression of viral antigen resulted in the spread of HSV to superficial cells and limited lateral spread to neighboring basal cells. The pathogenesis of HSV infection in these mice may serve as a model of the human recurrent epithelial disease in the progression of focal sites of infection and transfer from basal to superficial cells.

Schwann cells are removed from the spinal cord after effecting recovery from paraplegia.

Remyelination of the CNS is necessary to restore neural function in a number of demyelinating conditions. Schwann cells, the myelinating cells of the periphery, are candidates for this purpose because they have more robust regenerative properties than their central homologs, the oligodendrocytes. Although the ability of Schwann cells to remyelinate the CNS has been demonstrated, their capacity to enter the adult spinal cord in large numbers and effect functional recovery remains uncertain. We used cholera toxin B-subunit conjugated to saporin to demyelinate the rat lumbar spinal cord, remove macroglia, and produce paraplegia. After the removal of oligodendrocyte and astrocyte debris by invading macrophages, there was a spontaneous entry of Schwann cells into the spinal cord, along with axonal remyelination and concomitant functional recovery from paraplegia occurring within 75 d. The Schwann cells appeared to enter the dorsal funiculi via the dorsal root entry zone and the lateral funiculi via rootlets that had become adherent to the lateral spinal cord after the inflammation. In the following weeks, Schwann cell myelin surrounding central axons was progressively replaced by oligodendrocyte myelin without lapse in motor function. Our results show that endogenous Schwann cells can reverse a severe neurological deficit caused by CNS demyelination and enable later oligodendrocyte remyelination.

Vasoactive intestinal polypeptide in sacral primary sensory pathways in the cat.

Unmyelinated sensory axons in the sacral spinal cord may play a role in bladder reflexes under certain pathological conditions. Previous data suggested vasoactive intestinal polypeptide (VIP) might be contained exclusively in sensory C-fibers, some of which innervate the bladder. This study was undertaken to describe the morphology of these VIP fibers in the sacral cord of the cat. VIP immunoreactivity was confined to unmyelinated axons observed at several levels of the sensory pathway including the dorsal root ganglia, dorsal roots, Lissauer's tract, and the lateral collateral pathway. A combination of light and electron microscopic observations showed VIP-immunoreactive fibers with labeled varicosities and synaptic terminals in laminae I, IIo, V, VII, and X. VIP-immunolabeled varicosities had a mean diameter of 1.6 microm (range = 0.11-7.4 microm, S.D. = 1.01, n = 311) with a small percentage (8%) being relatively large (3-7.4 microm). VIP varicosities contained a mixture of small clear vesicles (CLV) and large dense core vesicles (LDV). Although most varicosities contained a moderate number of LDVs (14.86 LDVs/microm2), some varicosities contained a large number of LDVs, whereas others contained very few. Varicosities that possessed synaptic specializations were classed as terminals and were divided into three morphological classes. Two of these resembled Gray's Type I terminal, whereas a third was similar to the Gray's Type II terminal. There was no consistent relationship between vesicle content of the terminal and the type of synaptic contact it possessed. This study shows that in the sacral spinal cord of the cat, VIP terminals originate only from C-fibers, terminate primarily in laminae I and V, and exhibit a variety of morphologies consistent with heterogeneous origins and functions of the lower urinary tract.

Nitric oxide synthase containing neurons in the ventral lateral geniculate of the rat project to the optic pretectal nuclei.

We combined fluorescent tracing with immunohistochemistry to examine nitric oxide synthase (NOS) containing neurons in the rat ventral lateral geniculate nucleus (vLGN). NOS immunopositive neurons in vLGN had a similar appearance to previously described NADPH-d positive neurons. The majority (96%) of the NOS immunopositive neurons in vLGN projected to the pretectal nuclei and these represented 16% of all the vLGN neurons that project to the pretectal nuclei. No NOS immunopositive neurons projected to the superior colliculus. Co-localization of NADPH-d and gamma-aminobutyric acid (GABA) has been reported in vLGN neurons but we found few cells containing both NOS and GABA.

Noradrenergic input to nociceptive modulatory neurons in the rat rostral ventromedial medulla.

Within the rostral ventromedial medulla (RVM), there are two classes of putative pain modulation neurons: ON cells and OFF cells, which respectively burst or pause prior to withdrawal reflexes elicited by noxious stimulation. Alpha-adrenergic agonists injected into the RVM produce changes in the latency of spinal nocifensive reflexes and, when iontophoretically applied, alter the firing of RVM ON but not OFF cells. To provide further information about the contribution of norepinephrine to RVM neuron function, we analyzed the distribution of tyrosine hydroxylase immunoreactive (TH-ir) appositions upon RVM ON and OFF cells. In the lightly anesthetized rat, seven ON and five OFF cells were identified by changes in their discharge rate in relation to nociceptive withdrawal reflexes and were labeled by intracellular injection of neurobiotin. Sections containing labeled cells were visualized by using avidin conjugated to a Texas Red fluorophore. Tissue with labeled cells was subsequently processed for TH-ir by using a Bodipy fluorophore conjugated secondary antibody. The distribution of the Bodipy-labeled fibers and terminals upon the Texas Red-labeled neurons was mapped using a confocal laser-scanning microscope. All the labeled neurons exhibited close TH-ir appositions. Appositions were of two types: swellings and fibers. Although the numbers and density of appositions varied among the cells, there were no consistent differences that correlated with physiological properties. Thus the overall density of appositions for ON cells (29.0 +/- 22.2 x 10(4) microns2) did not differ significantly from that for OFF cells (25.4 +/- 22.2 x 10(4) microns2). Tyrosine hydroxylase immunoreactive (TH-ir) appositions upon ON and OFF cells varied with their location along the dorso-ventral axis with more ventral neurons having a greater density of TH-ir swelling-type appositions. In a separate study, TH-ir and dopamine-beta-hydroxylase-like immunoreactivity (DBH-ir) were mapped in the same sections by using confocal microscopy. Nearly 97% of the TH-ir profiles co-localized with DBH-ir. These observations provide evidence that both ON and OFF cells in the RVM are targeted by noradrenergic inputs.

Ultrastructural localization of nitric oxide synthase immunoreactivity in the cat ventrobasal complex.

This study describes the ultrastructural localization of nitric oxide synthase (NOS) immunoreactivity in the cat ventrobasal complex. NOS immunoreactivity was found in the cell bodies and dendrites of local circuit neurons and in vesicle-containing profiles. The vesicle-containing profiles could be divided into two classes, those of dendritic origin (presynaptic dendrite boutons) and those of axonal origin. The NOS labelled axon terminals varied in size and packing density and were principally located in the extra-glomerular neuropil. These boutons presented a range of morphologies and it was not possible to determine the probable source based on morphological criteria. The NOS immunoreactive presynaptic dendrite boutons were found both within and outside glomeruli and established both pre- and post-synaptic relationships with other elements. Post-embedding GABA immunocytochemistry showed that some NOS immunoreactive axonal boutons and presynaptic dendrites were also immunopositive for GABA. This finding suggests that some of the NOS labelled axonal boutons are of local circuit neuron origin. These results suggest that local circuit neurons in the cat ventrobasal complex might be involved in specific, short range interactions using GABA and longer, more global interactions using nitric oxide.

Dendritic arbors of neurons from different regions of the rat thalamic reticular nucleus share a similar orientation.

Neurons in different regions of the rat thalamic reticular nucleus were labeled with biotin dextran amine and reconstructed. When viewed in coronal section, some neurons had a radial dendritic tree while others had dorso-ventrally elongated arbors. When rotated, all the neurons had a planar, disc-shaped dendritic field with the dendrites orientated parallel to the long axis of the nucleus. We conclude that all thalamic reticular nucleus neurons have a similar dendritic morphology and orientation.

Transneuronal changes of the inhibitory circuitry in the macaque somatosensory thalamus following lesions of the dorsal column nuclei.

The inhibitory circuitry of the ventroposterolateral nucleus (VPL) of the macaque somatosensory thalamus was analyzed in normal animals and in those surviving for a few days or several weeks following a unilateral lesion of the cuneate nucleus, the source of medial lemniscal (ML) axons carrying information from the contralateral upper extremity. Inhibitory synaptic terminals in the VPL were defined as those that contain flattened or pleomorphic synaptic vesicles and that can be shown to be immunoreactive for gamma-aminobutyric acid (GABA). There are two types of these profiles: F axon terminals that arise from neurons of the thalamic reticular nucleus, and perhaps from VPL local circuit neurons (LCNs); and the dendritic appendages of LCNs that form presynaptic dendrites (PSDs). ML terminals normally have extensive synaptic interactions with PSDs but not with F axon terminals. Electron microscopic analyses revealed that cuneatus lesions resulted in a rapid loss of ML terminals and a statistically significant reduction in both F and PSD synaptic profiles. Confocal scanning microscopy also demonstrated a profound loss of GABA immunoreactivity in the deafferented VPL. These changes persisted for more than 20 weeks, without any evidence of reactive synaptogenesis of surviving sensory afferents or of inhibitory synapses. The changes in GABA circuitry are transneuronal, and the possible mechanisms that may underlie them are discussed. It is suggested that the altered GABAergic circuitry of the VPL in the monkey may serve as a model for understanding changes in somatic sensation in the human following peripheral or central deafferentation.

Nitric oxide synthase immunoreactivity distinguishes a sub-population of GABA-immunoreactive neurons in the ventrobasal complex of the cat.

This study describes the presence of nitric oxide synthase (NOS-ir) immunoreactive neurons in the thalamic ventrobasal complex of the cat. NOS-ir is co-localized with gamma-aminobutyric acid (GABA-ir) in a subset of small neurons identified as local circuit neurons in previous studies. The double labeled neurons are further identified by a larger soma diameter when compared to GABA-ir only neurons. All NOS-ir somata exhibit GABA-ir but none exhibit immunoreactivity to calbindin.

Architecture of individual dendrites from intracellularly labeled thalamocortical projection neurons in the ventral posterolateral and ventral posteromedial nuclei of cat.

This study provides quantitative descriptions of individual dendrites from electrophysiologically characterized and intracellularly labeled thalamocortical projection (TCP) neurons of the cat ventrobasal complex. One hundred nine dendrites from six ventral posterolateral (VPL) neurons and six ventral posteromedial (VPM) neurons were examined. Measurement of several parameters showed that the individual dendrites were very similar to each other in overall architecture even though they varied greatly in total length and number of dendritic branches. The mean path distance (length from soma to a dendritic tip) was very similar for all dendrites in each group (VPL or VPM) regardless of the number of branches found along the path distance. However, VPL dendrites had a longer mean path distance (VPL = 206 +/- 36 microns; n = 51) than VPM dendrites (VPM = 182 +/- 29 microns; n = 58; P < 0.001). For all dendrites there was a strong correlation between the stem dendrite diameter and the dendritic length, which allows the estimation of dendritic length from dendrite diameter. Analysis of dendritic scaling shows that branches higher than first order do not follow Rall's 3/2 power rule, so these neurons cannot be modeled using the equivalent cylinder approximation. The data add to the qualitative descriptions of cat ventrobasal (VB) TCP dendrites currently available and provide a basis for future comparative, developmental, and plasticity studies. Analysis shows that many parameters of cat VB TCP dendrites fall within a narrow range, suggesting that, regardless of differences in length or superficial appearance, these dendrites share a stable underlying architecture.

Analysis of gamma-aminobutyric acidergic synaptic contacts in the thalamic reticular nucleus of the monkey.

Gamma-aminobutyric acidergic (GABAergic) neurons in the thalamic reticular nucleus (TRN) spontaneously generate a synchronous bursting rhythm during slow-wave sleep in most mammals. A previous study at the electron microscopic level in cat anterior TRN has suggested that synchronous bursting activity could result from the large number of presumably GABAergic dendrodendritic synaptic contacts. However, little is known about the synaptology of the monkey thalamic reticular nucleus and whether it contains dendrodendritic contacts. To address this issue, we examined tissue obtained from Macaca fascicularis that was prepared for electron microscopy using postembedding techniques to demonstrate GABA immunoreactivity. Examination of the anterior (motor) and posterior (somatosensory) portions of the TRN disclosed the following: The majority of synaptic contacts (87.5% of 958) were formed by axon terminals showing no GABA immunoreactivity and making asymmetric synaptic contacts on dendrites or cell bodies. A further 6.4% of synaptic contacts was composed of GABA-immunoreactive presynaptic terminals making symmetric contacts with the dendrites of TRN neurons. The majority resembled the pleomorphic vesicle containing F-terminals seen in the dorsal thalamus and known to originate from axons of TRN. A subset or possible second class did not resemble any previously described class of GABA-immunoreactive terminals in the TRN. Both classes of these terminals making symmetric contacts may originate wholly or partially within the nucleus. There was one dendrodendritic synaptic contact and only a small number (3.2%) of axodendritic contacts with synaptic vesicles visible both pre- and postsynaptically. We conclude that dendrodendritic contacts are probably not responsible for the synchronized bursting neuronal activity seen in the slow-wave sleep of monkeys, and that, if TRN neurons are coupled synaptically, the most likely mechanism is through the synapses formed by recurrent axon collaterals of TRN neurons onto TRN dendrites.

Cell body and dendritic tree size of intracellularly labeled thalamocortical projection neurons in the ventrobasal complex of cat.

Fifteen thalamocortical projection (TCP) neurons from the adult cat ventrobasal complex (VB) were intracellularly labeled with horseradish peroxidase or neurobiotin and examined quantitatively. We find that cat TCP neurons share key morphological features and form one neuronal type. Previously reported variations in dendritic appearance cannot be supported by the present quantitative data. The number of dendrites varied between 4 and 13 (mean 9.1; +/- 4.0) and the total dendritic length of adult cat VB neurons varied between 9,421 and 19,646 microns (mean 13,120 microns; +/- 2,605). Linear regression analyses showed that soma diameter or cross-sectional area measurements provide a poor estimate for total dendritic length in TCP neurons. In contrast, the number of first order dendrites or the sum of first order dendrite diameters do provide a good estimate of overall TCP neuron size. This relationship is useful in predicting total dendritic length when it is not possible to reconstruct the entire dendritic tree. The mean dendritic path distance (distance from soma to the dendritic tip measured along the dendrite) was relatively constant for all neurons regardless of differences in total dendritic length or the number of branches that form the path distance.