Comparison of indirect peroxidase and avidin-biotin-peroxidase complex (ABC) immunohistochemical staining procedures for c-fos in rat brain.

c-Fos is the product of a gene expressed within neurons in the brain that serves as an anatomical marker of cellular activation. Immunohistochemical staining for c-fos allows a characterization of the effects of many different types of experimental manipulations on neuronal activity, making it a powerful technique for understanding brain, drug and behavior relationships. This study compared visualization of an anti-c-fos primary antibody in 40-µm-thick cryostat sections of formaldehyde-fixed rat brainstem using either a peroxidase enzyme-conjugated secondary antibody (indirect peroxidase) or the peroxidase-conjugated avidin-biotin complex (ABC) method. All sections were treated with H2 O2 to quench endogenous peroxidase enzyme and sodium borohydride to enhance permeability of the tissue and improve staining quality. Every other section was used to examine either the indirect peroxidase or the ABC method. Sections for the indirect peroxidase method were treated with Triton X-100 detergent to increase tissue permeability, goat serum to reduce non-specific binding of the secondary antibody and, in some cases, bovine serum albumin (BSA) to reduce non-specific binding of the primary antibody. Sections for the ABC method were treated with dilute normal serum, and avidin and biotin solutions and, in some cases BSA. Alternate sections were incubated for 72 h in either rabbit anti-c-fos primary antibody (1 : 20 000) or its vehicle (negative control). For the indirect peroxidase protocol, tissues were treated with peroxidase-conjugated goat anti-rabbit secondary antibody. For the ABC protocol, tissues were treated with biotinylated goat anti-rabbit secondary antibody and ABC peroxidase complex. All sections were reacted with 3,3'-diaminobenzadine (DAB) and H2 O2 , mounted and coverslipped. Both methods produced specific staining of c-fos-containing neurons, relative to the negative control sections. The indirect peroxidase protocol produced clear staining of c-fos-containing neurons, with very little background in the negative control sections. Staining for c-fos was enhanced using the ABC method in that c-fos stained neurons were darker and more clearly visible after shorter treatment with DAB. However, negative control sections showed a greater amount of non-specific staining with the ABC method. Thus, the ABC method was more sensitive but showed reduced specificity, with BSA treatment slightly reducing the level of non-specific staining. Overall, the ABC method produced better visualization and contrast of c-fos-containing neurons against the background color of the tissue.

Citalopram enhances B cell numbers in a murine model of morphine-induced immunosuppression.

Patients with chronic pain are often challenged with depression stemming from the long-term psychophysiological effects of their condition. Consequently, patients with chronic pain are often treated with morphine, which can induce immunosuppression, along with an antidepressant. The antidepressant citalopram (CTP; Sigma-Aldrich Chemical, St. Louis, MO, U.S.A.) is a serotonin reuptake inhibitor that is reported to have immunomodulatory effects. Thus, we investigated whether CTP administration impacted immunity in morphine-treated animals. Adult mice were pretreated for 7 days with either saline or CTP (10 or 30 mg/kg intraperitoneal injections twice daily), followed by subcutaneous implantation of a 25 mg morphine pellet for 48 hours. Spleen, thymus, and lymph nodes were harvested to analyze total cell numbers, relative lymphocyte populations, and lymphocyte function. In this study, CTP had no effect on either total cell counts or lymphocyte populations in the thymus. However, in the spleen, total splenocyte numbers in all CTP-treated animals displayed an increasing trend over saline-treated animals. Interestingly, although more cells were found in the spleen, distribution of splenic lymphocyte populations did not differ between treatments. Despite no increase in total cell number, a high dose of CTP (30 mg/kg) resulted in a significantly higher B cell population in the lymph nodes, while T cell and NK cell numbers were not different. CTP did not significantly reverse morphine-induced weight loss or splenic B cell antibody secretion in vitro. Additionally, CTP treatment demonstrated a slight but not significant increase in both splenic B and T cell mitogen-induced proliferation in vitro. In summary, CTP may have a specific potential in the attenuation of morphine's immunosuppressive effect by enhancing splenocyte numbers and lymph node B cell populations.

Voltammetric measurement of electrically evoked dopamine levels in the striatum of the anesthetized Syrian hamster.

Microdialysis measurements in the Syrian hamster clearly demonstrate a role for accumbal dopamine (DA) in female sexual behavior. However, large probe size and slow sampling rate prevent associating specific behaviors with DA changes in subregions of the heterogeneous nucleus accumbens. Fast-scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode (CFM), which affords millisecond temporal resolution at a micron-sized probe, could address these important issues. Mostly used in other rodents, e.g. rats and mice, this technique has not been applied to hamsters. The goal of the present study was to establish the measurement of DA in the nucleus accumbens of the anesthetized male Syrian hamster using FSCV at a CFM. For comparison, DA was simultaneously measured in the caudate-putamen. Stimulation of the medial forebrain bundle was used to elicit DA. Electrically evoked DA levels in both striatal regions were sensitive to location of the stimulating electrode and CFM, stimulation frequency, inhibition of DA uptake by cocaine and DA autoreceptor blockade by raclopride. Regional differences were observed for DA release and uptake parameters, and the effects of cocaine. Taken together, these results establish the measurement of electrically evoked DA levels in the hamster striatum using FSCV at a CFM.

The role of dopamine in reinforcement: changes in reinforcement sensitivity induced by D1-type, D2-type, and nonselective dopamine receptor agonists.

Dose-dependent changes in sensitivity to reinforcement were found when rats were treated with low, moderate, and high doses of the partial dopamine D1-type receptor agonist SKF38393 and with the nonselective dopamine agonist apomorphine, but did not change when rats were treated with similar doses of the selective dopamine D2-type receptor agonist quinpirole. Estimates of bias did not differ significantly across exposure to SKF38393 or quinpirole, but did change significantly at the high dose of apomorphine. Estimates of goodness of fit (r2) did not change significantly during quinpirole exposure. Poor goodness of fit was obtained for the high doses of SKF38393 and apomorphine. Decrements in absolute rates of responding were observed at the high dose of quinpirole and at the moderate and high doses of SKF38393 and apomorphine. Changes in r2 and absolute responding may be due to increases in stereotyped behavior during SKF38393 and apomorphine exposure that, in contrast to quinpirole, were distant from the response lever. The present data provide evidence that sensitivity to reward is affected more strongly by dopamine D1-like receptors rather than D2-like receptors, consistent with evidence from other studies investigating consummatory dopamine behavior and the tonic/phasic dopamine hypothesis.

Real-time processing of fast-scan cyclic voltammetry (FSCV) data using a field-programmable gate array (FPGA).

This paper reports the hardware implementation of a digital signal processing (DSP) unit for real-time processing of data obtained by fast-scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode (CFM), an electrochemical transduction technique for high-resolution monitoring of brain neurochemistry. Implemented on a field-programmable gate array (FPGA), the DSP unit comprises a decimation filter and an embedded processor to process the oversampled FSCV data and obtain in real time a temporal profile of concentration variation along with a chemical signature to identify the target neurotransmitter. Interfaced with an integrated, FSCV-sensing front-end, the DSP unit can successfully process FSCV data obtained by bolus injection of dopamine in a flow cell as well as electrically evoked, transient dopamine release in the dorsal striatum of an anesthetized rat.

Changes in sensitivity of response distributions to changing reinforcement ratios during exposure to ephedrine, caffeine, and ephedrine-caffeine combinations.

Changes in the sensitivity of response distributions to changes in reward distribution (reinforcer distribution sensitivity) were examined when rats were exposed to low and moderate doses of caffeine, ephedrine, and caffeine-ephedrine combinations. The data show significant decreases in sensitivity in response distributions to changes in reward schedule values during exposure to caffeine and ephedrine/caffeine combinations, whereas ephedrine alone resulted in overmatching comparable with baseline and NaCl conditions. Rats treated either with 3.0-mg/kg or 10.0-mg/kg doses of caffeine and all combinations of ephedrine at doses of 1.8 or 5.6 mg/kg with caffeine at 3.0 or 10.0 mg/kg showed reduced sensitivity in response distributions to differences in reinforcement schedule ratios. In contrast, when rats were exposed to ephedrine at 1.8 or 5.6 mg/kg, they maintained or increased the degree of overmatching. Although reinforcer distribution sensitivity was altered, drug exposure did not significantly affect the absolute rates of responding. Bias varied after exposure to caffeine, ephedrine, and their combinations, but not systematically. Finally, whereas the estimates of goodness of fit (r2) to the matching equation showed some decreases during drug exposure, these were neither statistically significant nor correlated with drug dose. These results suggest differential effects of ephedrine and caffeine on the sensitivity of response distributions to changes in reinforcement ratio distributions, with deleterious effects of caffeine and ephedrine/caffeine combinations.

Limbic pallidal adaptations following long-term cessation of dopaminergic transmission: lack of upregulation of dopamine receptor function.

Neurons in the ventral pallidum (VP) exhibit robust responding to activation of dopamine (DA) receptors of the D1 class. To determine if the VP adapts to chronic cessation of DA transmission, the present studies examined D1 receptor-mediated responses in the VP recorded extracellularly in chloral-hydrate anesthetized rats following destruction of DA neurons with 6-hydroxydopamine (6-OHDA) or long-term treatment with the D1 antagonist SCH23390. Indices of basal spiking (i.e., spontaneous firing rate and pattern) recorded 10-21 days after unilateral 6-OHDA treatment did not differ from controls. Moreover, DA depletion did not alter the proportion of VP neurons whose rate was enhanced with i.v. injections of the D1 agonist SKF38393, and the functional efficacy (Emax) and potency (ED50) were similar to controls. There also was no change in the direction of responses, the Emax or the ED50 measure of sensitivity (ECur50) to iontophoretic application of DA or SKF38393 in VP neurons. Forty-eight hours after 21 once-daily treatments with SCH23390, the number of [3H]SCH23390-labeled D1 receptors was increased in the striatum, but unchanged in the VP, globus pallidus, or septum. Accordingly, there was no functional upregulation of VP responses to i.v. SKF38393. Indeed, the proportion of SKF38393-sensitive neurons was decreased after chronic SCH23390. Distinguishing the VP from other forebrain regions, these findings indicate that basal spiking is not altered in the VP following chronic DA depletion, and that no upregulation of VP DA receptor function occurs following either dopaminergic lesions or chronic antagonism of D1 receptors.