In Situ Hybridization with Isotopic Riboprobes for Detection of Striatal Neuropeptide mRNA Expression After Dopamine Stimulant Administration - Drugs of Abuse: Neurological Reviews and Protocols - page 116

express primarily dopamine D 1 receptors and constitute the “direct” striatoni-
gral pathway (2 , 3) . In contrast, neurons containing the opioid neuropeptide
enkephalin predominantly express dopamine D 2 receptors and make up the
“indirect” striatopallidal circuitry (3 , 4) . A similar organization is present
in the nucleus accumbens ventral striatal region, with dynorphin primarily
expressed in neurons with D 1 and D 3 dopamine receptors (5 , 6) . Thus, elevation
of dopamine levels following administration of stimulant drugs will lead to
differential activation of dopamine receptors and subsequently modulation of
distinct striatal neuropeptide output circuits.
There are different methodologies by which striatal neuropeptidergic neurons
can be studied in relation to stimulant drug use. Alteration of gene expression is
an important mechanism through which long-term effects are maintained in the
brain and the compulsive, repeated use of addictive drugs implies impairments
at the level of gene expression. Measurements of mRNA levels can be achieved
through a variety of methodologies, but the technique of in situ hybridization
histochemistry (ISHH) has proven the most useful to detect mRNA expression
levels with intact anatomical integrity. Thus it is possible by the ISHH technique
to identify the discrete neuronal populations that express the preprodynorphin
(striatonigral) and preproenkephalin (striatopallidal) mRNAs. Using such
methodology, selective alterations of the opioid neuropeptide genes (increased
preprodynorphin and decreased preproenkephalin) were revealed in the striatum
of human cocaine users (7) . A similar up-regulation of the striatal preprodyn-
orphin gene has also been documented in a number of experimental rat models
following administration of cocaine (8-12) and amphetamines (13 , 14) that has
generated much attention on the dynorphin/D 1 /striatonigral neurons in the CNS
actions of stimulant drugs. The following details the ISHH procedure that can
be used to visualize the relative expression levels of the striatal neuropeptide
mRNAs following cocaine administration in the rat and human brain.
2. Materials
The following subheadings are organized as to the materials needed for (1)
brain tissue preparation and sectioning, (2) brain section pretreatment, (3) probe
preparation, (4) hybridization, (5) post-hybridization, and (6) autoradiography.
This outline corresponds to that provided in subsequent subheadings.
2.1. Brain Tissue Preparation and Sectioning
1. 2-Methylbutane (isopentane; Sigma).
2. Tissuetek OCT Compound (Histolab).
3. Superfrost microscope glass (Brain Research Laboratories).
4. SuperFrost Plus microscope glass (Menzel-Gläser).