Pain sensitivity following loss of cholinergic basal forebrain (CBF) neurons in the rat.

Flexion/withdrawal reflexes are attenuated by spinal, intracerebroventricular (ICV) and systemic delivery of cholinergic agonists. In contrast, some affective reactions to pain are suppressed by systemic cholinergic antagonism. Attention to aversive stimulation can be impaired, as is classical conditioning of fear and anxiety to aversive stimuli and psychological activation of stress reactions that exacerbate pain. Thus, in contrast to the suppressive effects of cholinergic agonism on reflexes, pain sensitivity and affective reactions to pain could be attenuated by reduced cerebral cholinergic activation. This possibility was evaluated in the present study, using an operant test of escape from nociceptive thermal stimulation (10 °C and 44.5 °C) before and after destruction of basal forebrain cholinergic neurons. ICV injection of 192 IgG-saporin produced widespread loss of basal forebrain cholinergic innervation of the cerebral cortex and hippocampus. Post-injection, escape from thermal stimulation was decreased with no indication of recovery for upto 19 weeks. Also, the normal hyperalgesic effect of sound stress was absent after ICV 192-sap. Effects of cerebral cholinergic denervation or stress on nociceptive licking and guarding reflexes were not consistent with the effects on operant escape, highlighting the importance of evaluating pain sensitivity of laboratory animals with an operant behavioral test. These results reveal that basal forebrain cholinergic transmission participates in the cerebral processing of pain, which may be relevant to the pain sensitivity of patients with Alzheimer's disease who have prominent degeneration of basal forebrain cholinergic neurons.

Neuropeptide Y receptor-expressing dorsal horn neurons: role in nocifensive reflex and operant responses to aversive cold after CFA inflammation.

The spinal Neuropeptide Y (NPY) system is a potential target for development of new pain therapeutics. NPY and two of its receptors (Y1 and Y2) are found in the superficial dorsal horn of the spinal cord, a key area of nociceptive gating and modulation. Lumbar intrathecal injection of (NPY) is antinociceptive, reducing hyper-reflexia to thermal and mechanical stimulation, particularly after nerve injury and inflammation. We have also shown that intrathecal injection of the targeted cytotoxin, Neuropeptide Y-sap (NPY-sap), is also antinociceptive, reducing nocifensive reflex responses to noxious heat and formalin. In the present study, we sought to determine the role of dorsal horn Y1R-expressing neurons in pain by destroying them with NPY-sap and testing the rats on three operant tasks. Lumbar intrathecal NPY-sap (1) reduced Complete Freund's Adjuvant (CFA)-induced hyper-reflexia on the 10°C cold plate, (2) reduced cold aversion on the thermal preference and escape tasks, (3) was analgesic to noxious heat on the escape task, (4) reduced the CFA-induced allodynia to cold temperatures experienced on the thermal preference, feeding interference, and escape tasks, and (5) did not inhibit or interfere with morphine analgesia.

Galanin receptor-expressing dorsal horn neurons: role in nociception.

Galanin, along with enkephalins and neuropeptide Y, has been hypothesized to negatively modulate nociception in the superficial dorsal horn of the spinal cord. In the present study, we sought to determine the role of presumably excitatory dorsal horn galanin receptor-expressing neurons in nociception by selectively destroying GalR1-expressing superficial dorsal horn interneurons using lumbar intrathecal injections of the targeted cytotoxin, galanin-saporin (Gal-sap). Lumbar intrathecal injection of Gal-sap (500 ng) reduced immunoperoxidase staining for GalR1 in the superficial dorsal horn without affecting primary afferent neurons in lumbar dorsal root ganglia. Lumbar intrathecal Gal-sap also: 1--reduced nocifensive reflex responding on the thermal plate at 0.3 °C, 44 °C, and 47 °C; 2--increased hot side occupancy in a thermal preference task (15 °C vs 45 °C); and, 3--decreased escape from 44 °C and 47 °C, but not 20 °C. Thus, similar to lesions of mu opiate receptor-expressing dorsal horn interneurons, selective destruction of GalR1-expressing superficial dorsal horn neurons produces heat hypo-algesia, likely due to loss of GalR1-expressing excitatory interneurons leading to reduced activation of nociceptive projection neurons in response to aversive heat. These results are different than those seen with intrathecal neuropeptide Y-saporin and suggest the potential value of selectively targeting GalR1-expressing dorsal horn neurons to control pain.

Neuropeptide Y receptor-expressing dorsal horn neurons: role in nocifensive reflex responses to heat and formalin.

Lumbar intrathecal injection of neuropeptide Y (NPY) is antinociceptive, particularly in models of nerve injury and inflammation. Intrathecal NPY does not alter nociception in mice null for the Y1 neuropeptide Y receptor (Y1R) and these mice show enhanced nocifensive reflex responses to aversive thermal, mechanical, visceral and chemical stimuli. Y1R and NPY receptor type 2 (Y2R) are present in the spinal dorsal horn presynaptically on primary afferent, and possibly interneuron terminals, but only Y1R is found postsynaptically on dorsal horn neurons. In the present study, we sought to assess the anatomic effects of lumbar intrathecal disulfide conjugate of neuropeptide Y and saporin (NPY-sap) and to determine the role of Y1R-expressing dorsal horn neurons in nocifensive responses to aversive thermal and chemical stimulation. Lumbar intrathecal injection of NPY-sap was used to selectively destroy Y1R-expressing lumbar dorsal horn neurons followed by testing nocifensive reflex responses on the hotplate and after hind-paw formalin injection. NPY-saporin decreased superficial dorsal horn staining for Y1R, but not neurokinin-1 receptor, mu opiate receptor or NPY peptide, and had no effect on Y1R cell counts in fourth lumbar spinal segment dorsal root ganglia. Loss of Y1R-expressing dorsal horn neurons was associated with increased first response latencies on the 44 degrees C hotplate and reduced total time rats spent licking and guarding hind paws during 600 s trials at 44 degrees C or 200 s trials at 47 degrees C. First hind-paw response latencies to high intensity phasic stimulation at 52 degrees C were unaffected. NPY-sap also reduced formalin-induced nocifensive behaviors during both interphase and phase II. These data demonstrate that selective destruction of Y1R-expressing superficial dorsal horn neurons, probably excitatory interneurons and/or projection neurons, reduces nocifensive reflex responses, particularly to activation of C nociceptors, and suggest a possible role for Y1R-expressing dorsal horn neurons in pain.

Anti-nociceptive effects of selectively destroying substance P receptor-expressing dorsal horn neurons using [Sar9,Met(O2)11]-substance P-saporin: behavioral and anatomical analyses.

Lumbar intrathecal injections of substance P-saporin (SP-sap) destroy dorsal horn neurons that express the neurokinin-1 receptor (NK-1R) resulting in decreased responses to a range of noxious stimuli and decreased hyperalgesia and allodynia. Forebrain injections of SP-sap produce considerable non-specific damage raising some concern about use of this toxin in vivo. The more stable and selective substance P congener, [Sar9,Met(O2)11]substance P coupled to saporin (SSP-sap) produces much more selective forebrain lesions at significantly lower doses. The present study sought to determine the anatomic and nocifensive behavioral effects of lumbar intrathecal injections of the more precisely targeted SSP-sap. On the basis of loss of lamina I NK-1R staining, lumbar intrathecal SSP-sap was seven times more potent than SP-sap and produced no loss of NK-1R expressing neurons in deeper laminae (III-VI or X). Transient decreases in hotplate responding occurred at 44 degrees C and 47 degrees C but not 52 degrees C during the first 3 weeks after SSP-sap injection with return to baseline by 4 weeks. Operant escape responses were reduced at 0.3 degrees C, 44 degrees C and 47 degrees C for at least 4 months. In the formalin test, SSP-sap also was about seven times more potent than SP-sap in reducing phase two behavior in both female Long Evans and male Sprague-Dawley rats. Both SSP-sap and SP-sap reduced formalin-induced FOS expression in deep and superficial laminae of the L4 dorsal horn in parallel with the reduction in phase 2 behavior. In summary, SSP-sap is highly effective in destroying lamina I NK-1R expressing neurons, without loss of deep NK-1R neurons. The behavioral effects of SSP-sap are similar to SP-sap suggesting that the antinociceptive effects of both toxins are indeed due to selective loss of NK-1R neurons in lamina I. SSP-sap is an attractive agent for possible treatment of chronic pain.

Destruction of midbrain dopaminergic neurons by using immunotoxin to dopamine transporter.

1. The ability to target specific neurons can be used to produce selective neural lesions and potentially to deliver therapeutically useful moieties for treatment of disease. In the present study, we sought to determine if a monoclonal antibody to the dopamine transporter (anti-DAT) could be used to target midbrain dopaminergic neurons. 2. The monoclonal antibody recognizes the second, large extracellular loop of DAT. The antibody was conjugated to the "ribosome-inactivating protein"; saporin, and stereotactically pressure microinjected into either the center of the striatum or the left lateral ventricle of adult, male Sprague-Dawley rats. 3. Local intrastriatal injections produced destruction of dopaminergic neurons in the ipsilateral substantia nigra consistent with suicide transport of the immunotoxin. Intraventricular injections (i.c.v.) produced significant loss of dopaminergic neurons in the substantia nigra and ventral tegmental area bilaterally without evident damage to any other aminergic structures such as the locus coeruleus and raphe nuclei. To confirm the anatomic findings, binding of [3-H]mazindol to DAT in the striatum and midbrain was assessed using densitometric analysis of autoradiograms. Anti-DAT-saporin injected i.c.v. at a dose of 21 microg, but not 8 microg, produced highly significant decreases in mazindol binding consistent with loss of the dopaminergic neurons. 4. These results show that anti-DAT can be used to target midbrain dopaminergic neurons and that anti-DAT-saporin may be useful for producing a lesion very similar to the naturally occurring neural degeneration seen in Parkinson's disease. Anti-DAT-saporin joins the growing list of neural lesioning agents based on targeted cytotoxins.

Intrathecal substance p-saporin attenuates operant escape from nociceptive thermal stimuli.

Destruction of neurons in the superficial dorsal horn that express substance P receptor (NK-1R) has been reported to block development of behavioral hypersensitivity following peripheral sensitization of nociceptors. Baseline sensitivity was not altered in these rat models that assessed innate reflex responses (i.e. hind-paw withdrawal to thermal or mechanical stimulation). In the present study, we evaluated effects of intrathecal substance P-saporin (SP-sap), a toxin selective for cells expressing NK-1R, on operant escape responses of rats to thermal stimulation. For comparison, lick/guard reflex testing was performed. Injection of a modest dose (175 ng) of SP-sap into the lumbar subarachnoid space produced a partial loss of lamina I/II NK-1R-expressing dorsal horn neurons but did not affect NK-1R-expressing neurons in deeper laminae. Lick/guard responses to 0.3, 44 or 47 degrees C were not affected after SP-sap treatment, but escape responses to these temperatures were significantly attenuated. Three hours after application of mustard oil to the dorsal surface of both hind paws, escape from 44 degrees C was enhanced for controls but not SP-sap-treated rats. Lick/guard responses were enhanced by mustard oil for both SP-sap and control animals. Administration of morphine (1.0 mg/kg, s.c.) before testing decreased escape responding at 47 degrees C for both controls and SP-sap rats. Thus, partial loss of NK-1R-expressing neurons in the superficial dorsal horn attenuated thermal nociceptive sensitivity and prevented secondary hyperalgesia when studied with an operant algesia assay, in contrast to innate reflexes which were less sensitive to modification by intrathecal SP-sap.

Colocalization of mu-opioid receptors and activated G-proteins in rat cingulate cortex.

Anterior cingulate cortex (ACC) has a role in pain processing, however, little is known about opioid system organization and actions. This rodent study defines opioid architecture in the perigenual and midcingulate divisions of ACC, relates mu-opioid receptor binding and G-protein activation, and localizes such binding to afferent axons with knife-cut lesions and specifically to noradrenergic terminals with immunotoxin lesions (anti-dopamine beta-hydroxylase-saporin; anti-DBH-saporin). [(3)H]Tyr-D-AlaGly-MePhe-Gly-ol (DAMGO) binding was highest in perigenual areas 32 and 24 with a peak in layer I. Midcingulate area 24' and posterior cingulate area 29 had overall lower binding in each layer. In contrast, DAMGO-stimulated [(35)S]guanosine-5'-O-(gamma-thio)-triphosphate (GTPgammaS) binding in area 24' was similar to that in area 24, whereas area 29 had low and homogeneous binding. Undercut lesions reduced [(3)H]DAMGO binding in all layers with the greatest loss in layer I (-65%), whereas DAMGO-stimulated [(35)S]GTPgammaS binding losses occurred in only layers I-III. Anti-DBH-saporin reduced [(3)H]DAMGO binding in layer I of area 24; DAMGO-stimulated [(35)S]GTPgammaS binding was unchanged in areas 24' and 29. Correlation analysis of receptor and G-protein activation before and after undercut lesions suggested there were a greater number of DAMGO receptor sites for each G-protein on axons, than on somata and proximal dendrites. Finally, perigenual and midcingulate cortices have different opioid architectures due to a higher proportion of mu-opioid receptors expressed by afferent axons in areas 24 and 32.

Lack of effect of moderate Purkinje cell loss on working memory.

192 immunoglobulin G-saporin (192-sap) is an immunotoxin which targets the cholinergic basal forebrain after injection into either the ventricular system or the parenchyma of the rat brain. When injected by the i.c.v. route, 192-sap kills some cerebellar Purkinje cells in addition to its more extensive killing of the cholinergic basal forebrain. Behaviorally, i.c.v. injections of 192-sap result in impaired performance in a variety of experimental paradigms of learning and memory including a working memory task in the radial maze. The current study examined the contribution, if any, of immunotoxin-induced Purkinje cell loss to impaired performance in the radial maze. To meet this aim, we used i.c.v. injection of another immunotoxin, OX7-saporin (OX7-sap), at a dose that produced Purkinje cell loss of similar extent to that produced by i.c.v. 192-sap. We then compared these OX7-sap-injected rats with 192-sap-injected rats in a radial maze working memory task. We found a working memory impairment only in the 192-sap-injected rats. These data show that moderate Purkinje cell loss alone is insufficient to impair working memory. Furthermore, the data are consistent with the idea that the working memory deficit observed in 192-sap-injected animals is likely due to lesioning of the cholinergic basal forebrain.

Targeted toxins.

Molecular neurosurgery can be used to make selective neural lesions by targeting cytotoxins to specific populations of neurons based on their common expression of a particular surface molecule. The targeted toxins employed in this unit consist of a targeting moiety (vector) and an effector moiety (cytotoxin). In all cases discussed in this unit, the cytotoxic moiety is an enzyme that catalytically inactivates the large ribosomal subunit, irreversibly inhibiting protein synthesis and resulting in cell death. These toxins appear to kill in an all-or-none fashion, indicating that one molecule of free cytotoxin in the cytoplasm of a cell is sufficient to kill the cell. Three general molecular neurosurgery protocols are presented in this unit. The first describes suicide transport, which refers to the use of targeted toxins to make anatomically restricted lesions based on retrograde axonal transport of the toxin. The second involves immunolesioning and uses anti-neuronal immunotoxins to make type-selective and anatomically restricted lesions. The final protocol uses neuropeptide-toxin conjugates to selectively destroy neurons expressing the receptor for the specific neuropeptide.

Neuronal lesioning with axonally transported toxins.

Axonally transported toxins can be used to make selective lesions of the nervous system. Collectively, these techniques are termed 'molecular neurosurgery' because they exploit the surface molecular identity of neurons to selectively destroy specific types of neurons. Suicide transport, is anatomically selective but not type-selective. The most widely used suicide transport agents are the toxic lectins (ricin, volkensin) and the immunotoxin, OX7-saporin. The toxic lectins and saporin are ribosome inactivating proteins that irreversibly inhibit protein synthesis. The toxic lectins have binding subunits but saporin requires a targeting vector to gain entrance into cells. Immunolesioning uses monoclonal anti-neuronal antibodies to deliver saporin selectively into neurons that express a particular target surface antigen. Neuropeptide-saporin conjugates selectively destroy neurons expressing the appropriate peptide receptors. Notable experimental uses of these agents include analysis of the function of the cholinergic basal forebrain (192-saporin) and pain research (anti-DBH-saporin, substance P-saporin). It is likely that more immunolesioning and neuropeptide-toxin conjugates will be developed in the near future.

Enhancement of cortical plasticity by behavioral training in acetylcholine-depleted adult rats.

Trimming all whiskers except two on one side of an adult rat's face results in cortical plasticity in which the spared whiskers, D2 and one D-row surround whisker (either D1 or D3), evoked responses containing more spikes than the response evoked by the cut whisker (called whisker pairing plasticity). Previously we have reported that acetylcholine (ACh) depletion in cortex prevents surround D-row whisker plasticity from developing within the barrel cortex. In this study we examined whether the animal's active use of its two intact whiskers can restore some aspects of plasticity in the ACh-depleted cortex. To achieve this goal, ACh was depleted from barrel field cortex, and 14 days after the depletion surgery, whiskers were trimmed and animals were trained on a whisker-dependent gap crossing task. After 7 days of training, animals were anesthetized with urethan and prepared for single-unit recording. Training the ACh-depleted, whisker-paired animals resulted in a significant enhancement of responses to paired surround whiskers: the D-paired whisker-evoked response contained more spikes than the D-cut evoked response. We conclude that training whisker paired rats has a positive impact on response properties of neurons in S1 cortex, even in ACh-depleted animals.

Entering through the doors of perception: characterization of a highly selective Substance P receptor-targeted toxin.

Perception of external stimuli is often mediated through the activity of a G protein-coupled receptor in response to its ligand. Receptor-mediated internalization allows the insertion of toxins that cause the elimination of receptor-expressing neurons. Using this technique new information on systems biology can be discovered and with this, new therapeutics developed.

Molecular heterogeneity of protein kinase C expression in human ventricle.

OBJECTIVE: Although activation of protein kinase C (PKC) modulates the function of normal cardiac myocytes and likely plays a role in the pathogenesis of cardiomyopathic disease states, the molecular basis of PKC expression in human ventricle has not been examined in detail. METHODS: We have performed Western analysis and immunohistochemistry on explanted human cardiac tissue from nondiseased and diseased specimens using isoform-specific antibodies directed against all known PKC isozymes. RESULTS: In homogenates from left and right ventricle, all isoforms except PKC-gamma and theta were detected by immunoblotting, with confirmation using a second antibody directed against a different epitope when possible. For PKC-betaII, delta, and epsilon, data indicated that these isoforms were variably phosphorylated in vivo, resulting in multiple bands during immunoblotting. Because of potential antibody cross-reactivity, reverse transcriptase polymerase chain reaction (RT-PCR) was performed which confirmed expression of PKC-alpha, betaI, and zeta. Immunohistochemistry demonstrated that all isoforms detected in ventricular homogenate by Western analysis could be localized to cardiac myocytes. From a methodologic standpoint, significant degradation of PKC isoforms could be demonstrated when samples were either frozen or allowed to remain at room temperature, compared to immediate subcellular fractionation. CONCLUSIONS: These findings indicate that the PKC expression in human ventricular myocytes is remarkably diverse, with multiple conventional, novel, and atypical isoforms present, and highlight the importance of sample preparation in comparative studies of PKC isoform expression.

Preferential destruction of cerebellar Purkinje cells by OX7-saporin.

Saporin, a plant toxin derived from Saponaria officinalis, disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex. OX7 is a mouse monoclonal antibody directed against the Thy-1.1 receptor that is differentially expressed on subpopulations of central nervous system neurons. Disulfide conjugation of OX7 to saporin permits delivery of saporin to target neurons. OX7-saporin was used to study the behavioral and morphological consequences of selective destruction of cerebellar Purkinje cells which abundantly express the Thy-1.1 antigen. Male Sprague-Dawley rats received bilateral intraventricular injections of 1- or 2 microg OX7-saporin or 8 microl artificial CSF. Rats were tested for behavioral changes 1 week before and 1, 2, and 8 weeks post-treatment. OX7-saporin treatment resulted in dose- and time-dependent changes in motor performance, activity, and negative geotaxis, but did not affect foot splay. Following behavioral testing, cerebellar sections were prepared for microscopic examination and the pattern of Purkinje cell loss was determined in anatomically matched sections. OX7-saporin induced dose-dependent death of Purkinje cells, particularly in the anterior and superior portions of cerebellar folia 1-6 and folium 9. Other brain regions appeared largely unaffected. Data suggest that intraventricular injection of rats with OX7-saporin is an effective model with which to examine the consequences of Purkinje cell destruction.

Impaired acquisition of a Morris water maze task following selective destruction of cerebellar purkinje cells with OX7-saporin.

Spatial learning in the Morris water maze task is believed to be dependent on an intact hippocampal system. However, evidence from human studies and animal experiments suggests a potential cerebellar involvement in spatial processing, place learning, and other types of 'higher-order' cognition. In order to investigate this possibility, intraventricular injections (ICV) of the anti-neuronal immunotoxin OX7-saporin were used to selectively destroy cerebellar Purkinje cells, without affecting other brain areas believed to be critically involved in spatial learning and memory. Bilateral ICV injections of 2 microg OX7-saporin (4 microg total) in adult male rats produced substantial loss of Purkinje cells (56%) throughout the cerebellum without affecting hippocampal morphology or biochemical indices of cholinergic, serotonergic, or catecholaminergic function in the hippocampus, frontal cortex, or striatum. ICV OX7-saporin significantly impaired acquisition and performance of the standard Morris water maze task (though the impairment was less severe than reported in earlier studies that used alternate lesion methods or mutant mice species), but did not alter performance on the cued version of the task, or locomotor activity. In addition, lesioned animals spent significantly less time in the target quadrant on probe trial days 4 and 7 and the average distance to target scores (ADT) were significantly greater than controls on those days. Swim speed was not affected. Based on the specificity of the behavioral and neurobiological alterations, these data support the hypothesis that the cerebellum is involved in spatial processing and place learning.

Immunolocalization of the cocaine- and antidepressant-sensitive l-norepinephrine transporter.

Norepinephrine (NE) transporters (NETs) constitute the primary mechanism for inactivation of synaptically released NE, are targets for multiple antidepressants and psychostimulants, and have been reported to be deficient in affective and autonomic disorders. Although the regional distribution of NETs has been defined through synaptosomal transport and autoradiographic approaches, NET protein expression has yet to be characterized fully in the central nervous system (CNS). We identified a cytoplasmic NET epitope (amino acids 585-602) and corresponding antibody (43411) that permits cellular localization of endogenous NET expression in the CNS and periphery. In the adult rat brain, NET labeling was confined to noradrenergic neuronal somata, axons, and dendrites, including extensive arborizations within the hippocampus and cortex, but was absent from epinephrine- and dopamine-containing neurons. Intracerebroventricular anti-dopamine beta-hydroxylase/saporin, a treatment that destroys a majority of noradrenergic neurons and their projections, validated the specificity of the 43411 antibody. At the level of light microscopy, 43411 labeling colocalized with the axonal markers syntaxin, synaptophysin, and SNAP-25. Indirect immunofluorescence revealed a nonuniform pattern of NET expression along axons, particularly evident within sympathetic fibers of the vas deferens, reflecting a high degree of spatial organization of NE clearance. NET labeling in somata was intracellular and absent from plasma membranes. Among nonneuronal cells, glial cells lacked NET immunoreactivity, whereas CNS ependymal cells were an unexpected site of labeling. NET immunoreactivity was also evident in a subset of adrenal chromaffin cells where labeling appeared to be predominantly associated with intracellular vesicles. Initial ultrastructural evaluation via preembedding immunogold techniques also revealed substantial cytoplasmic NET immunoreactivity in axon terminals within the prelimbic prefrontal cortex, consistent with postulates of regulated trafficking controlling neurotransmitter clearance. NET visualization should be of significant benefit in evaluating neuronal injury resulting from chronic drug exposure and in disease states.