Migration of molecules in the brain : focus on the extracellular space and the cerebrospinal fluid

Three different molecules and three different experimental models were used to study the migration of molecules in the rat brain: a neurotransmitter- dopamine, an extracellular market- mannitol, and a neuropeptide beta-endorphin. Two models in vivo were used to study the diffusion and migration of molecules: dual-probe rnicrodialysis in the striatum, and direct intrastriatal microinjections followed by cerebrospinal fluid sampling from the cisterna magna. One model ex vivo was used to study the functional relevance of molecular migration: immunohistochemis try of dopamine terminal and D1 receptor mismatches in the amygdala.

Using dual-probe microdialysis, long distance diffusion of 3H-dopamine and 3H-mannitol was investigated in control and 6-OHDA-lesioned anaesthetized male rats. Physiological levels of 3Hdopamine were continuously delivered and recovered 1 mm away during 5 hours, and the dialysates were separated by HPLC into dopamine and metabolite fractions. The transfer profiles of the total amount of 3H-dopamine-derived compounds described the overall effect of cellular uptake, metabolism and clearance of the compounds into the microcirculation. These profiles reached steady state levels after 1-2 hours, generating an equilibrium between delivery and removal from the extracellular space. The half time to reach the steady state levels was lower in 6OHDA treated rats compared to controls. The metabolites 3H-DOPAC and 3H-HVA were found also in 6OHDA-lesioned rats, reflecting the presence of metabolizing enzymes MAO and COMT at sites other than within dopaminergic terminals. The 3H-dopamine-derived transfer profiles were compared to the diffusion of 3H-mannitol. The diffusion halftime values of 3H-mannitol were lower than those of 3H-dopamine and its metabolites in both control and 6-OHDA lesioned rats, implying that extracellular compounds diffuse faster than molecules that possess both extracellular and intracellular transport routes. No unmetabolized 3Hdopamine, but a substantial amount of unidentified metabolites, were detected.

The diffusion of 3H-mannitol in the striatum was further studied by assessing its diffusion in brain compared to diffusion in a dilute agar gel. The data was analyzed according to a mathematical model designed for dualprobe microdialysis. The parameters volume fraction alpha [the ratio of the extracellular space volume to the total tissue volume], the tortuosity lambda [the increase in path length], and a clearance rate constant kappa [the overall removal of tracer from the extracellular space], were obtained from the mathematical model. Theoretical expressions were derived for the spatial concentration distribution in the brain and the temporal dialysate concentrations of the infused tracer. A program was developed to fit the experimental data by fine-tuning the relevant parameters. The model could accurately predict the diffusion behavior of 3H-mannitol in dual-probe microdialysis experiments.

In order to study the migration of larger molecules, such as peptides, direct intraparenchymal administration and sampling of CSF from the cisterna magna was used. We investigated whether microinjected exogenous rat beta-endorphin could migrate from the striatum into the lateral ventricle and the general CSF circulation. The beta-endorphin content in the CSF was quantified by radio-immunoassay, and showed high levels of betaendorphin-like immunoreactive JR) material. Mass spectrometry analysis of the CSF revealed that betaendorphin was primarily present as an intact peptide, and not as proteolytic cleavage fragments. betaendorphin-IR was found in nerve cell bodies at distant regions from the injection site, such as the globus pallidus. Confocal laser microscopy showed that beta-endorphin-IR and mu-opioid receptors colocalized at the plasma membrane. These findings support the idea that beta-endorphin may diffuse within the brain parenchyma and reach the CSF in the ventricular system and thereby activate distant opioid receptors.

To put the migration of molecules in the brain in a functional perspective, a transmitter-receptor mismatch study was performed using double immunofluorescence histochemistry of dopamine terminals and D1 receptors in the amygdala. The amygdala is involved in elicitation and learning of fear-related behaviors, and dopamine transmission in selected subregions of the amygdala was hypothesized to be able to influence and regulate the generation of fear. The intercalated islands of the amygdala are D1-rich GABAergic neurons interposed between the major input and output structures of the amygdala. In the rostromedial and caudal parts of the main intercalated island, there was only a minor dopaminergic innervation, suggesting that dopamine participates in local short-distance migration in these regions. Slow dopaminergic volume transmission in these parts may have a role in a tonic excitatory modulation of the GABAergic neurons, that inhibit the central amygdaloid nucleus and thereby contributing to inhibition of fear related behavior. In contrast, the rostrolateral main island and paracapsular islands showed a high degree of DA innervation. Thus, a fast synaptic/perisynaptic dopaminergic transmission in these parts may have a role in allowing a more rapid elicitation of fear related behaviors, through disinhibition of the GABA inputs from the rostromedial and caudal parts, projecting to the central amygdaloid nucleus. In summary, these findings give valuable insights into the volume transmission theory.

List of scientific papers

I. Hoistad M, Kehr J, Andbjer B, Jansson A, Fuxe K (2000). "Intracerebral infusion of H-dopamine and H-mannitol in the striatum of halothane-anaesthetized male rats. A dual-probe microdialysis study of long-distance diffusion. " Eur J Neurosci 12(7): 2505-14
https://pubmed.ncbi.nlm.nih.gov/10947826

II. Hoistad M, Chen KC, Nicholson C, Fuxe K, Kehr J (2002). "Quantitative dual-probe microdialysis: evaluation of [3H]mannitol diffusion in agar and rat striatum. " J Neurochem 81(1): 80-93
https://pubmed.ncbi.nlm.nih.gov/12067240

III. Chen KC, Hoistad M, Kehr J, Fuxe K, Nicholson C (2002). "Quantitative dual-probe microdialysis: mathematical model and analysis. " J Neurochem 81(1): 94-107
https://pubmed.ncbi.nlm.nih.gov/12067242

IV. Hoistad M, Samskog J, Jacobsen K, Olsson A, Brodin E, Hansson HA, Fuxe K (2004). "Beta-endorphin microinjected intrastriatally in the rat is detected in the CSF with radioimmunoassay and mass-spectrometry." (Manuscript)

V. Fuxe K, Jacobsen KX, Hoistad M, Tinner B, Jansson A, Staines WA, Agnati LF (2003). "The dopamine D1 receptor-rich main and paracapsular intercalated nerve cell groups of the rat amygdala: relationship to the dopamine innervation. " Neuroscience 119(3): 733-46
https://pubmed.ncbi.nlm.nih.gov/12809694