Increased acetylcholine and glutamate efflux in the prefrontal cortex following intranasal orexin-A (hypocretin-1)

Abstract

Orexin/hypocretin neurons of the lateral hypothalamus and perifornical area are integrators of physiological function. Previous work from our laboratory and others has shown the importance of orexin transmission in cognition. Age-related reductions in markers of orexin function further suggest that this neuropeptide may be a useful target for the treatment of age-related cognitive dysfunction. Intranasal administration of orexin-A (OxA) has shown promise as a therapeutic option for cognitive dysfunction. However, the neurochemical mechanisms of intranasal OxA administration are not fully understood. Here, we use immunohistochemistry and in vivo microdialysis to define the effects of acute intranasal OxA administration on: (i) activation of neuronal populations in the cortex, basal forebrain, and brainstem and (ii) acetylcholine (ACh) and glutamate efflux in the prefrontal cortex (PFC) of Fischer 344/Brown Norway F1 rats. Acute intranasal administration of OxA significantly increased c-Fos expression, a marker for neuronal activation, in the PFC and in subpopulations of basal forebrain cholinergic neurons. Subsequently, we investigated the effects of acute intranasal OxA on neurotransmitter efflux in the PFC and found that intranasal OxA significantly increased both ACh and glutamate efflux in this region. These findings were independent from any changes in c-Fos expression in orexin neurons, suggesting that these effects are not resultant from direct activation of orexin neurons. In total, these data indicate that intranasal OxA may enhance cognition through activation of distinct neuronal populations in the cortex and basal forebrain and through increased neurotransmission of ACh and glutamate in the PFC.

Keywords: acetylcholine; cognition; hypocretin; intranasal; microdialysis; orexin.

Fig. 1

General experimental timeline. Day 0 represents the day of arrival of each batch of animals at the University of South Carolina School of Medicine vivarium. Handling and habituation of the rats began the following day. After one week, animals were assigned to either the c-Fos expression or in vivo microdialysis experimental groups. For the c-Fos experiments animals received a week of daily habituation to intranasal saline. On the test day (Day 15) each rat received intranasal OxA or saline and was euthanized 2 hr later. Processing of brain tissue, immunohistochemistry, cell counts and data analysis began on Day 16 and proceeded until complete for each batch of rats used for c-Fos analysis. For the in vivo microdialysis experiments, guide cannula surgery was performed during Days 8–10. After several days of recovery, animals received a week of daily habituation to intranasal saline. The first microdialysis session was performed on Day 19 and the second session on Day 21. Half of the rats received OxA in the first session and saline in the second session; the order was reversed for the other half of the rats. On the day following the second microdialysis session (Day 22) the rats were euthanized, their brains processed for histochemical verification of probe placement, and HPLC analysis of dialysate samples began.

Fig. 2

Neuronal activation (c-Fos expression density) in cortical brain regions following intranasal OxA administration. (a) Single-labeled c-Fos density for animals treated with intranasal saline (vehicle; PrLC, AIC, VOC, n=8 rats) or intranasal OxA (50 µl, 100 µM; PrLC and AIC, n=8 rats; VOC, n=7 rats). Treatment with intranasal OxA resulted in increased c-Fos expression in neurons of PrLC, AIC, and VOC compared to vehicle treated controls (b) Diagram indicating the approximate location (black-outlined square) within the PrLC for which c-Fos counts and photomicrographs were obtained. (c) Single-label immunohistochemistry for c-Fos shown as blue/black punctate nuclei following acute treatment with intranasal saline. (d) Single-label immunohistochemistry for c-Fos following acute treatment with intranasal OxA. A clear increase in c-Fos expression density is seen in the OxA treatment group (d) compared to vehicle controls (c). Abbreviations: OxA, orexin-A; PrLC, prelimbic prefrontal cortex; AIC, agranular insular cortex; VOC, ventral orbital cortex. Scale bar represents approximately 100 µm (c, d). Error bars represent SEM. **p<0.01, *p<0.05

Fig. 3

Activation of PV-positive GABAergic interneurons in cortical brain regions after intranasal OxA administration. (a) Percentage of double-labeled (c-Fos/PV) neurons relative to the total number of PV neurons within the PrLC, VOC, AIC, ILC, MOC, PirC, and Cl after treatment with intranasal saline (vehicle; all regions, n=8 rats) or intranasal OxA (50 µl, 100 µM; PrLC, AIC, ILC, PirC, and Cl, n=8 rats; VOC and MOC, n=7 rats). Intranasal OxA significantly increased the percentage of dual-labeled PV neurons in the PrLC and VOC. Other cortical regions including the AIC, ILC, MOC, and PirC, as well as the subcortical claustrum, did not show significant differences between treatment groups. (b) Schematic indicating the approximate location (black-outlined square) within the PrLC where c-Fos/PV counts and photomicrographs were obtained. (c) Typical dual-label immunohistochemistry for c-Fos (blue/black) and PV (brown) neurons within the PrLC (arrows). Abbreviations: PV, parvalbumin; OxA, orexin-A; PrLC, prelimbic prefrontal cortex; VOC, ventral orbital cortex; AIC, agranular insular cortex; ILC, infralimbic prefrontal cortex; MOC, medial orbital cortex; PirC, piriform cortex; Cl, Claustrum. Scale bar represents approximately 100 µm (c). Error bars represent SEM. ***p<0.001, **p<0.01

Fig. 4

Activation of cholinergic neurons in the basal forebrain by intranasal OxA administration. (a) Percentage of double-labeled (c-Fos/ChAT) neurons relative to the total number of ChAT positive neurons within the VDBB, VP/SI, MS, and HDBB after treatment with intranasal saline (vehicle; all regions, n=8 rats) or intranasal OxA (50 µl, 100 µM; all regions, n=8 rats). Intranasal OxA significantly increased the percentage of dual-labeled ChAT neurons in the VDBB and VP/SI compared to vehicle treated controls. (b) Schematic indicating the approximate location (black-outlined square) within the VDBB where c-Fos/ChAT counts and photomicrographs were obtained. (c) Typical dual-label immunohistochemistry for c-Fos (blue/black) and ChAT (brown) neurons within the VDBB (arrows). Abbreviations: OxA, orexin-A; ChAT, choline acetyltransferase; VDBB, vertical limb of the diagonal band; VP/SI, ventral pallidum/substantia innominata; MS, medial septum; HDBB, horizontal limb of the diagonal band. Scale bar represents approximately 100 µm (c). Error bars represent SEM. ***p<0.001, **p<0.01

Fig. 5

Effect of intranasal OxA administration on ACh and glutamate efflux in the PFC. (a) Intranasal OxA (50 µl, 100 µM; n=10 rats) treatment after baseline collections (arrow) significantly increased ACh efflux compared to intranasal vehicle (saline; n=10 rats). A significant increase in ACh efflux was observed from time points five through nine versus vehicle (b) Intranasal OxA (arrow; n=10 rats) also significantly increased glutamate efflux within the prefrontal cortex compared to treatment with intranasal saline. The significant effect lasted from time points five through eight versus vehicle treatment. (c) Diagram indicating the approximate probe placement within the PFC for each of the animals that underwent microdialysis. Typical probe placement in the PFC is indicated on AChE background-stained section from an animal that underwent two microdialysis sessions. Abbreviations: PFC, prefrontal cortex; ACh, acetylcholine; in, intranasal; OxA, orexin-A. Error bars represent SEM. **p<.01, *p<.05

Fig. 6

Localization of a green-fluorescent tagged OxA peptide in the brain after intranasal administration. (a) Typical distribution of the fluorescent-tagged OxA peptide (50 µl, 500 µM) in the PrLC after intranasal administration. Green fluorescence indicates the appearance of the labeled peptide in the PrLC. (b) Schematic indicating the approximate location (black-outlined square) within the PrLC where fluorescence photomicrographs were obtained. (c) Typical distribution of the fluorescent-tagged OxA peptide in the SpTrN. (d) Schematic indicating the approximate location (black-outlined square) within the SpTrN where fluorescence photomicrographs were obtained. Abbreviations: OxA, orexin-A; PrLC, prelimbic prefrontal cortex; SpTrN, spinal trigeminal nucleus. Scale bar approximately 100 µm.