Comprehensive Physiology Wiley Online Library

Anatomical Markers of Activity in Hypothalamic Neurons

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Abstract

The scientific community has searched for years for ways of examining neuronal tissue to track neural activity with reliable anatomical markers for stimulated neuronal activity. Existing studies that focused on hypothalamic systems offer a few options but do not always compare approaches or validate them for dependence on cell firing, leaving the reader uncertain of the benefits and limitations of each method. Thus, in this article, potential markers will be presented and, where possible, placed into perspective in terms of when and how these methods pertain to hypothalamic function. An example of each approach is included. In reviewing the approaches, one is guided through how neurons work, the consequences of their stimulation, and then the potential markers that could be applied to hypothalamic systems are discussed. Approaches will use features of neuronal glucose utilization, water/oxygen movement, changes in neuron‐glial interactions, receptor translocation, cytoskeletal changes, stimulus‐synthesis coupling that includes expression of the heteronuclear or mature mRNA for transmitters or the enzymes that make them, and changes in transcription factors (immediate early gene products, precursor buildup, use of promoter‐driven surrogate proteins, and induced expression of added transmitters. This article includes discussion of methodological limitations and the power of combining approaches to understand neuronal function. © 2020 American Physiological Society. Compr Physiol 10:549‐575, 2020.

Keywords: pathway; physiological genomics; excitation; neurophysiology

Figure 1. Figure 1. (A) Autoradiogram of 14C 2‐deoxyglucose (2‐DG) on a slice containing the suprachiasmatic nuclei (SCN) that was obtained during the subjective day; (B) a slice taken from an identical area obtained during the subjective night; and (C) a Nissl‐stained section of the same area as B verifying the presence of the SCN. Note that the signal, while clear, does not easily enable resolution of separate cells. Adapted, with permission, from Newman GC, et al., 1992 118. Copyright 1992, Society for Neuroscience.
Figure 2. Figure 2. (A) fMRI changes (red) in the posterior hypothalamus that correlated with social dominance in macaque monkeys. (B) Correlation of the level of activation with behavioral measures of social dominance. Adapted, with permission, from Noonan MP, et al., 2014 120.
Figure 3. Figure 3. Example of glial‐neuron changes in the supraoptic nucleus of (A) an normal female rat with a clear astrocytic investment (as) around the neuron and (B) a lactating rat in which multiple synapses (arrows) are observed when the glial investment of the neuron is withdrawn. Adapted, with permission, from Theodosis DT, et al., 2004 151.
Figure 4. Figure 4. Indirect evidence for the presence of gap junctions between neurons (A and C) through detection of coupled neurons after a fluorescent dye is injected into only one of the two neurons. (B and D) Cells that appear to lack gap junction communication. Adapted, with permission, from Hatton GI and Yang QZ, 2001 56.
Figure 5. Figure 5. Dendrodendritic interactions in GnRH neurons. Intercellular bridges are seen between GnRH dendrites. (A) Examples of dendritic processes from a rhesus monkey whose bridges between immunoreactive GnRH neurons appear as fine ladders (Hoffman G, unpublished data). (B) Example of two intercellular bridges across GnRH neurons in a rat brain (red circle). (C) Dendritic bundling between two (arrowheads) or three dendrites (arrows) of GnRH neurons labeled with either green fluorescent protein (colored green) or injected biocytin (colored red or yellow). Adapted, with permission, from Campbell RE, et al., 2009 14.
Figure 6. Figure 6. Example of μ‐opiate receptor (MOR) translocation in response to estrogen treatment. (A) Ovariectomized female rat (OVX); (B) OVX rat treated with estradiol benzoate (50 μg, EB). This approach is used as evidence that a receptor ligand has affected neuronal activity. Adapted, with permission, from Eckersell CB, et al., 1998 36. Copyright 1998 Society for Neuroscience.
Figure 7. Figure 7. Spines on kisspeptin neurons of the arcuate nucleus are abundant in ovariectomized animals (A–C) in contrast to ovariectomized animals given estrogen replacement (D). The relationship of the spine patterns to firing dynamics of the neurons leads to the use of spine morphology as a marker of the neuron's activity. Adapted, with permission, from Cholanian M, et al., 2015 20.
Figure 8. Figure 8. Changes in the expression of the cytoskeletal protein, activity‐regulated cytoskeletal protein (Arc/Arg3.1), reflect changes in neuron activity. The figure shows induced ARC/Arg3.1 expression in the suprachiasmatic nucleus in response to lights on in a mouse at 0, 60, 120, 180, and 300 min after lights on. Adapted, with permission, from Nishimura M, et al., 2003 119.
Figure 9. Figure 9. Vasopressin mRNA in neurons of the supraoptic nucleus in a control normonatremic animal (A and B) and after 7 days of hyponatremia. Hyponatremia, a treatment that reduces firing of vasopressin neurons, reduces markedly mRNA expression in the neurons. C and D are higher magnifications of the cells. The bar graph (E) compares the mRNA levels determined from in situ hybridization to those of solution hybridization analysis for vasopressin (Exon C). Hyponatremia significantly decreased mRNA for vasopression (**p < 0.001). The magnitude of suppression with the two detection methods was similar. Adapted, with permission, from Hoffman G, et al., 1995 63.
Figure 10. Figure 10. Tyrosine hydroxylase (TH) RNA levels in continuously suckling rats (0 h) and at 1.5, 6, 12, and 24 h after pup removal reveal initial nuclear sites (heteronuclear RNA) in the first hours after pup removal, and then gradual reexpression of mature mRNA in the cell cytoplasm to levels seen in cycling rats at the later times. Adapted, with permission, from Berghorn KA, et al., 2001 6.
Figure 11. Figure 11. Staining of the protein prohormone for vasopressin and oxytocin is low under baseline conditions (A), but after hypernatremia (B) is strikingly elevated in the supraoptic nucleus. Adapted, with permission, from Verbalis JG, et al., 1991 155.
Figure 12. Figure 12. Examples of the use of promoter‐driven flag expression. a) Example of use of the vasopressin promoter to drive expression of green fluorescent protein (GFP) to enable detection of changes in vasopressin's synthetic activity in chronically salt‐loaded rats. Compared to controls (A–C), expression of the GFP in the SON, PVN, and internal zone of the median eminence of salt‐loaded rats is strikingly elevated (D–F). Adapted, with permission, from Suzuki H, et al., 2009 146. b) Changes in a flag linked to the activity‐depended cytoskeletal protein ARC in the parvocellular PVN (circle) in a control rat (A), a rat exposed to an acute restraint stress (B), or a rat exposed to repeated restraint stress (C). (D) A rat exposed to an immune challenge (lipopolysaccharide, LPS) 3 h earlier. (E) An LPS animal after 6 h. (F) An animal stimulated by both repeated restraint and LPS. (G) A higher magnification of (F) revealing the GFP flag for ARC. (H) Labeling of the same section for CRH. (I) The double labeling of ARC (green) and CRH. Adapted, with permission, from Grinevich V, et al., 2009 53.
Figure 13. Figure 13. Aberrant expression of surrogate proteins. The fact that all cells of the body have the same DNA can result in expression of a surrogate protein in cells that would normally not express that protein. The likely explanation for this is that the insertion of a transgene under a defined promoter lacks sequences that would normally suppress the protein. The figure illustrates examples of aberrant expression of GnRH‐directed green fluorescent protein in populations of neurons that normally would not express GnRH in the lateral hypothalamus (A, black box) and in the dorsal septal area (B, white box). Hoffman G, unpublished data.
Figure 14. Figure 14. Direct assay of mRNA expression. (A) Striking changes in the mRNA for tyrosine hydroxylase detected by radioactive in situ hybridization technology in cycling, lactating rats, and lactating rats whose pups were removed for 24 h. Reused, with permission, from Wang HJ, et al., 1993 158. That neural activity determines the suppression of TH mRNA during suckling in the arcuate nucleus is illustrated after blocking nipples on only one side of the body as illustrated in B. In C, TH mRNA is upregulated back to baseline levels in the arcuate nucleus only on the side contralateral to the nipple blockade. Like other somatosensory paths to the thalamus, the course from the nipples to the thalamus also crosses. Reused, with permission, from Berghorn KA, et al., 2001 6.
Figure 15. Figure 15. Example of a dose‐response in the induction of Fos (left figures) in oxytocin neurons (right figures) after graded treatment with cholecystokinin. Reused, with permission, from Verbalis JG, et al., 1991 156.
Figure 16. Figure 16. Example of the monitoring of fluorescent Ca++ sensitive dyes in electrically stimulated suprachiasmatic nucleus neurons from slice preparations. Reused, with permission, from Irwin RP and Allen CN, 2013 73.
Figure 17. Figure 17. Example of the induction of galanin (black) in GnRH neurons following a surge in the GnRH system. Interestingly, the GnRH in the neurons (brown) is not markedly changed. Hoffman G, unpublished data. Activity dependence of this phenomenon is seen when the GnRH system's surge is delayed for 24 h and delays the galanin expression.
Figure 18. Figure 18. Examples of the changes in CRH hnRNA and that of vasopressin in neurons of the parvocellular PVN after a single or repeated restraint stress. The upper panels (A–D) present CRH hnRNA, and the lower panels (E–H) vasopressin hnRNA. (A) control naïve rat, (B) single restraint, (C) basal CRH expression before repeated restraint, and (D) CRH hnRNA after 14 restraint episodes. Note that CRH hnRNA is elevated by acute restraint but is suppressed after repeated stress (either before or after the last restraint). (E) The expression of vasopressin hnRNA is low in the parvocellular PVN (MP) of naïve rats. However, after a single or multiple episodes of restraint, the vasopressin hnRNA is elevated (F–H). Reused, with permission, from Aguilera G, et al., 2008 1.
Figure 19. Figure 19. Effective use of timing differences in expression of hnRNA versus cytoplasmic mRNA for tyrosine hydroxylase following administration of a glutamate agonist. (A) Control; (B) 1 h after delivery of NMDA. Hoffman G, unpublished data.


Figure 1. (A) Autoradiogram of 14C 2‐deoxyglucose (2‐DG) on a slice containing the suprachiasmatic nuclei (SCN) that was obtained during the subjective day; (B) a slice taken from an identical area obtained during the subjective night; and (C) a Nissl‐stained section of the same area as B verifying the presence of the SCN. Note that the signal, while clear, does not easily enable resolution of separate cells. Adapted, with permission, from Newman GC, et al., 1992 118. Copyright 1992, Society for Neuroscience.


Figure 2. (A) fMRI changes (red) in the posterior hypothalamus that correlated with social dominance in macaque monkeys. (B) Correlation of the level of activation with behavioral measures of social dominance. Adapted, with permission, from Noonan MP, et al., 2014 120.


Figure 3. Example of glial‐neuron changes in the supraoptic nucleus of (A) an normal female rat with a clear astrocytic investment (as) around the neuron and (B) a lactating rat in which multiple synapses (arrows) are observed when the glial investment of the neuron is withdrawn. Adapted, with permission, from Theodosis DT, et al., 2004 151.


Figure 4. Indirect evidence for the presence of gap junctions between neurons (A and C) through detection of coupled neurons after a fluorescent dye is injected into only one of the two neurons. (B and D) Cells that appear to lack gap junction communication. Adapted, with permission, from Hatton GI and Yang QZ, 2001 56.


Figure 5. Dendrodendritic interactions in GnRH neurons. Intercellular bridges are seen between GnRH dendrites. (A) Examples of dendritic processes from a rhesus monkey whose bridges between immunoreactive GnRH neurons appear as fine ladders (Hoffman G, unpublished data). (B) Example of two intercellular bridges across GnRH neurons in a rat brain (red circle). (C) Dendritic bundling between two (arrowheads) or three dendrites (arrows) of GnRH neurons labeled with either green fluorescent protein (colored green) or injected biocytin (colored red or yellow). Adapted, with permission, from Campbell RE, et al., 2009 14.


Figure 6. Example of μ‐opiate receptor (MOR) translocation in response to estrogen treatment. (A) Ovariectomized female rat (OVX); (B) OVX rat treated with estradiol benzoate (50 μg, EB). This approach is used as evidence that a receptor ligand has affected neuronal activity. Adapted, with permission, from Eckersell CB, et al., 1998 36. Copyright 1998 Society for Neuroscience.


Figure 7. Spines on kisspeptin neurons of the arcuate nucleus are abundant in ovariectomized animals (A–C) in contrast to ovariectomized animals given estrogen replacement (D). The relationship of the spine patterns to firing dynamics of the neurons leads to the use of spine morphology as a marker of the neuron's activity. Adapted, with permission, from Cholanian M, et al., 2015 20.


Figure 8. Changes in the expression of the cytoskeletal protein, activity‐regulated cytoskeletal protein (Arc/Arg3.1), reflect changes in neuron activity. The figure shows induced ARC/Arg3.1 expression in the suprachiasmatic nucleus in response to lights on in a mouse at 0, 60, 120, 180, and 300 min after lights on. Adapted, with permission, from Nishimura M, et al., 2003 119.


Figure 9. Vasopressin mRNA in neurons of the supraoptic nucleus in a control normonatremic animal (A and B) and after 7 days of hyponatremia. Hyponatremia, a treatment that reduces firing of vasopressin neurons, reduces markedly mRNA expression in the neurons. C and D are higher magnifications of the cells. The bar graph (E) compares the mRNA levels determined from in situ hybridization to those of solution hybridization analysis for vasopressin (Exon C). Hyponatremia significantly decreased mRNA for vasopression (**p < 0.001). The magnitude of suppression with the two detection methods was similar. Adapted, with permission, from Hoffman G, et al., 1995 63.


Figure 10. Tyrosine hydroxylase (TH) RNA levels in continuously suckling rats (0 h) and at 1.5, 6, 12, and 24 h after pup removal reveal initial nuclear sites (heteronuclear RNA) in the first hours after pup removal, and then gradual reexpression of mature mRNA in the cell cytoplasm to levels seen in cycling rats at the later times. Adapted, with permission, from Berghorn KA, et al., 2001 6.


Figure 11. Staining of the protein prohormone for vasopressin and oxytocin is low under baseline conditions (A), but after hypernatremia (B) is strikingly elevated in the supraoptic nucleus. Adapted, with permission, from Verbalis JG, et al., 1991 155.


Figure 12. Examples of the use of promoter‐driven flag expression. a) Example of use of the vasopressin promoter to drive expression of green fluorescent protein (GFP) to enable detection of changes in vasopressin's synthetic activity in chronically salt‐loaded rats. Compared to controls (A–C), expression of the GFP in the SON, PVN, and internal zone of the median eminence of salt‐loaded rats is strikingly elevated (D–F). Adapted, with permission, from Suzuki H, et al., 2009 146. b) Changes in a flag linked to the activity‐depended cytoskeletal protein ARC in the parvocellular PVN (circle) in a control rat (A), a rat exposed to an acute restraint stress (B), or a rat exposed to repeated restraint stress (C). (D) A rat exposed to an immune challenge (lipopolysaccharide, LPS) 3 h earlier. (E) An LPS animal after 6 h. (F) An animal stimulated by both repeated restraint and LPS. (G) A higher magnification of (F) revealing the GFP flag for ARC. (H) Labeling of the same section for CRH. (I) The double labeling of ARC (green) and CRH. Adapted, with permission, from Grinevich V, et al., 2009 53.


Figure 13. Aberrant expression of surrogate proteins. The fact that all cells of the body have the same DNA can result in expression of a surrogate protein in cells that would normally not express that protein. The likely explanation for this is that the insertion of a transgene under a defined promoter lacks sequences that would normally suppress the protein. The figure illustrates examples of aberrant expression of GnRH‐directed green fluorescent protein in populations of neurons that normally would not express GnRH in the lateral hypothalamus (A, black box) and in the dorsal septal area (B, white box). Hoffman G, unpublished data.


Figure 14. Direct assay of mRNA expression. (A) Striking changes in the mRNA for tyrosine hydroxylase detected by radioactive in situ hybridization technology in cycling, lactating rats, and lactating rats whose pups were removed for 24 h. Reused, with permission, from Wang HJ, et al., 1993 158. That neural activity determines the suppression of TH mRNA during suckling in the arcuate nucleus is illustrated after blocking nipples on only one side of the body as illustrated in B. In C, TH mRNA is upregulated back to baseline levels in the arcuate nucleus only on the side contralateral to the nipple blockade. Like other somatosensory paths to the thalamus, the course from the nipples to the thalamus also crosses. Reused, with permission, from Berghorn KA, et al., 2001 6.


Figure 15. Example of a dose‐response in the induction of Fos (left figures) in oxytocin neurons (right figures) after graded treatment with cholecystokinin. Reused, with permission, from Verbalis JG, et al., 1991 156.


Figure 16. Example of the monitoring of fluorescent Ca++ sensitive dyes in electrically stimulated suprachiasmatic nucleus neurons from slice preparations. Reused, with permission, from Irwin RP and Allen CN, 2013 73.


Figure 17. Example of the induction of galanin (black) in GnRH neurons following a surge in the GnRH system. Interestingly, the GnRH in the neurons (brown) is not markedly changed. Hoffman G, unpublished data. Activity dependence of this phenomenon is seen when the GnRH system's surge is delayed for 24 h and delays the galanin expression.


Figure 18. Examples of the changes in CRH hnRNA and that of vasopressin in neurons of the parvocellular PVN after a single or repeated restraint stress. The upper panels (A–D) present CRH hnRNA, and the lower panels (E–H) vasopressin hnRNA. (A) control naïve rat, (B) single restraint, (C) basal CRH expression before repeated restraint, and (D) CRH hnRNA after 14 restraint episodes. Note that CRH hnRNA is elevated by acute restraint but is suppressed after repeated stress (either before or after the last restraint). (E) The expression of vasopressin hnRNA is low in the parvocellular PVN (MP) of naïve rats. However, after a single or multiple episodes of restraint, the vasopressin hnRNA is elevated (F–H). Reused, with permission, from Aguilera G, et al., 2008 1.


Figure 19. Effective use of timing differences in expression of hnRNA versus cytoplasmic mRNA for tyrosine hydroxylase following administration of a glutamate agonist. (A) Control; (B) 1 h after delivery of NMDA. Hoffman G, unpublished data.
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Gloria E. Hoffman. Anatomical Markers of Activity in Hypothalamic Neurons. Compr Physiol 2020, 10: 549-575. doi: 10.1002/cphy.c170021