Stimulation of lateral hypothalamic glutamate and acetylcholine efflux by nicotine: implications for mechanisms of nicotine-induced activation of orexin neurons

Abstract

The hypothalamus is a prominent target of nicotine action. We have previously shown that acute systemic nicotine treatment induces Fos expression in the lateral hypothalamus and perifornical area (LH/PFA), with orexin/hypocretin neurons being particularly responsive. However, the neurochemical correlates of acute nicotine treatment in the LH/PFA have not been described. Anatomical studies have revealed that this area receives afferents from cholinergic, glutamatergic, and GABAergic telencephalic brain regions, suggesting a potential role for these neurotransmitters in mediating the hypothalamic component of nicotine effects on homeostatic phenomena, such as arousal and appetite. Here, we used in vivo microdialysis to determine the effect of acute systemic or local nicotine on glutamate, acetylcholine, and GABA efflux in the LH/PFA of rats. Local administration of nicotine significantly increased acetylcholine and glutamate, but not GABA, in the LH/PFA. Thus, we further tested the role of afferent sources of glutamate and acetylcholine in mediating acute nicotine-induced activation of orexin neurons by unilaterally lesioning the prefrontal cortex or basal forebrain cholinergic regions. Lesioned animals showed reduced Fos-positive orexin neurons following nicotine treatment. These data suggest that both acetylcholine and glutamate may mediate the effects of acute nicotine on the activity of hypothalamic neurons, including orexin/hypocretin cells. Changes in cholinergic or glutamatergic transmission in this region with chronic nicotine may contribute to long-term alterations in functions mediated by LH/PFA neurons, including feeding and arousal.

Figure 1

Mean raw basal LH/PFA efflux values (uncorrected for probe recovery) for ACh (A), glutamate (B) and GABA (C) during the three systemic or local nicotine treatment sessions. Basal efflux was not significantly altered for any of the three neurotransmitters as a function of microdialysis session or route of nicotine treatment. D. Coronal hemisection schematic of probe placement (modified from Paxinos and Watson, 1997) in LH/PFA. E. Representative low-magnification photomicrograph of probe tract in the hypothalamus. F. Higher-magnification of the area outlined in (E) demonstrating the microdialysis probe tract surrounded by orexin-immunoreactive neurons (arrows). Abbreviations: 3v, third ventricle; fx, fornix; scale bar in (F) represents approximately 500μm. Error bars represent SEM.

Figure 2

Effect of nicotine administration on ACh efflux in the LH/PFA. A. Systemic nicotine (0, 0.4 mg/kg or 2.0 mg/kg) did not affect ACh efflux in the LH/PFA. B. Local nicotine (100 μM) increased ACh efflux whereas the higher concentration of nicotine (2.0 mM) significantly decreased ACh efflux during the eighth collection interval. Nicotine reached the LH/PFA during the 6th-9th collection intervals. * P<0.05 vs. vehicle and 2.0 mM nicotine. # P<0.05 vs. 2.0 mM nicotine. Error bars represent SEM.

Figure 3

Effect of nicotine administration on glutamate efflux in the LH/PFA. A. Systemic nicotine (0, 0.4 mg/kg or 2.0 mg/kg) did not affect glutamate efflux in the LH/PFA. B. Local nicotine (2.0 mM) increased glutamate efflux whereas the lower concentration of nicotine (100 μM) significantly decreased glutamate efflux during the seventh collection interval. * P<0.05 vs. vehicle. # P<0.05 vs. 100 μM nicotine. Error bars represent SEM.

Figure 4

Effect of nicotine administration on GABA efflux in the LH/PFA: GABA efflux was not significantly altered by either systemic (A) or local (B) nicotine. Error bars represent SEM.

Figure 5

Activation of orexin neurons by nicotine following prefrontal cortical lesions. A. Double-labeled (Fos/orexin) neurons on the injected side relative to the contralateral hemisphere in control (Sham) or ibotenic acid (Ibo) PFC-lesioned animals treated acutely with systemic vehicle (Veh) or nicotine (Nic). Ibotenic acid lesions resulted in a significant reduction in nicotine-activated orexin neurons ipsilateral to the lesion site. This attenuation was seen in both medial and lateral sectors of the LH/PFA. *P < 0.05 vs. Sham-Nic. B. Dual-label immunohistochemistry for orexin and Fos following nicotine (2 mg/kg) treatment in a sham-lesioned animal. Numerous double-labeled cells are seen in the perifornical area (arrows). C. Dual-label immunohistochemistry following acute nicotine in an ibotenic acid PFC-lesioned animal. Some double-labeling is observed (arrows) but less than in (B). D. Cresyl violet-stained hemisection from the medial PFC of a sham-lesioned animal. E. Cresyl violet staining in the medial PFC of an ibotenic acid-lesioned animal. The lesioned area is demarcated by extensive gliosis. F. Schematic indicating typical lesion extent following ibotenic acid infusions of the PFC. Abbreviations: fx, fornix; fmi, forceps minor of the corpus callosum; cg1, anterior cingulate cortex; pl, prelimbic cortex; il, infralimbic cortex. Scale bars: approximately 100 μm (B/C); approximately 500 μm (D/E).

Figure 6

Activation of orexin neurons by nicotine following basal forebrain cholinergic lesions. A. Double-labeled (Fos/orexin) neurons on the injected side relative to the contralateral hemisphere in control (Sham) or 192 IgG-saporin (SAP) basal forebrain-lesioned animals treated acutely with systemic vehicle (Veh) or nicotine (Nic). 192 IgG-saporin lesions resulted in a significant reduction in nicotine-activated orexin neurons ipsilateral to the lesion site. This attenuation was seen in both medial and lateral sectors of the LH/PFA. *P < 0.05 vs. Sham-Nic. B. 192 IgG-saporin (SAP) treatment resulted in a greater than 80% loss of ChAT-immunoreactive cells in the basal forebrain relative to the non-lesioned hemisphere. No significant loss of cholinergic cells was seen in the sham-lesioned animals. *P < 0.05 vs. Sham. C. Immunohistochemistry for ChAT in the basal forebrain of a unilaterally-lesioned animal. The non-infused (left) hemisphere is characterized by the presence of numerous ChAT-immunoreactive cells in basal forebrain regions encompassing the substantia innominata, ventral pallidum and horizontal limb of the diagonal band of Broca, as indicated by the rectangle. The 192 IgG-saporin-infused (right) hemisphere is largely devoid of ChAT-immunoreactive neurons. Abbreviations: ac, anterior commissure; 3v, third ventricle. Scale bar equals approximately 1 mm.

Figure 7

Effect of PFC ibotenic acid or basal forebrain 192 IgG-saporin lesions on vGlut1 or vAChT immunoreactivity in the LH/PFA. A. Ibotenic acid lesions of the PFC reduced the number of apparent appositions between vGlut1-immunoreactive varicosities and orexin neurons. B & C. Double-label immunohistochemistry for vGlut1 (dark puncta) and orexin A (light brown cell somata) from a sham (B) or ibotenic acid-treated rat (C). Arrows indicate points of apparent appositional contact. D. The cholinergic immunotoxin 192 IgG-saporin in the basal forebrain reduced the number of apparent appositions between vAChT-immunoreactive varicosities and orexin neurons. E & F. Double-label immunohistochemistry for vAChT (dark puncta) and orexin neurons (light brown somata) from a sham (E) or 192 IgG-saporin-treated (F) rat. Arrows indicate apparent appositional contacts. Scale bars, approximately 20 μm. *P < 0.05 vs. sham-lesioned group.