Abstract
Prolactin (PRL) is a pleotropic hormone, exerting multifaceted effects on various body tissues. Its secretion from the anterior pituitary is primarily regulated by dopamine (DA) released from tuberoinfundibular dopaminergic (TIDA) neurons located in the hypothalamic arcuate nucleus. During lactation, when PRL levels need to rise, this mechanism undergoes significant remodeling, including changes in the neurochemistry and morphology of the TIDA neurons, leading to reduced DA synthesis. Release of DA from TIDA neurons is also significantly decreased during lactation, although the cellular mechanisms mediating this modification of TIDA neuron behaviour are poorly understood. Given that DA release patterns of TIDA neurons in male mice primarily rely on their activity properties (Stagkourakis et al., 2019, 2020), we hypothesised that the activity properties of TIDA neurons in rats undergo adaptive modifications to mediate changes in DA release during lactation.
We utilised Ca2+ imaging to monitor the network and single-cell activity patterns of TIDA neurons in virgin male, virgin female, lactating females and post-lactating rats. Stereotaxic injection of a Cre-inducible virus carrying GCaMP6s into TH-Cre rats allowed the specific expression of this Ca2+ indicator in TIDA neurons.
Initial recordings of TIDA neurons derived from virgin males and females revealed a high degree of activity synchronisation within the TIDA network of both. However, at the single cell level, TIDA neurons in females exhibited higher oscillation frequency and rhythmicity than those in males. In contrast, TIDA neurons from lactating animals displayed activity dynamics that were marked by a low degree of activity synchronisation, with TIDA neurons oscillating at low or absent rhythmicity. Those with remaining rhythmicity oscillated at a heterogeneous set of frequencies. Notably, most of these lactational adaptations were unique to the TIDA system and not observed in another population of dopaminergic neurons in the rostral arcuate nucleus. To mimic weaning, when circulatory PRL levels are restored to pre-lactational levels, we also performed recordings of TIDA neurons in dams separated from their pups for 2 and 10 days. These recordings revealed a gradual restoration of these neurons to pre-lactational activity patterns. These findings indicate that the TIDA neurons are highly adaptable in their activity profiles and elucidate the role of these profiles in governing DA release.
To examine the mechanisms responsible for the TIDA neuronal activity, we conducted Ca2+ imaging recordings in the presence of pharmacological blockers of connexin 36 (Cx36) gap junctions, or of fast ionotropic glutamatergic and GABAergic transmission. Blocking Cx36 gap junctions diminished activity synchronisation and oscillation rhythmicity in TIDA neurons of both male and female animals. However, Cx36 gap junction blockade had no effect on the activity properties of TIDA neurons in lactating animals suggesting a possible downregulation of these junctions during lactation. Inhibiting fast ionotropic glutamatergic and GABAergic synaptic transmission had a sexually dimorphic effect where TIDA neurons in males were unaffected, while those in virgin females experienced an increase in oscillation frequency and a decrease in oscillation rhythmicity. Inhibiting these synapses in lactating animals however had no effect on TIDA neuron activity, implying a potential downregulation of these synapses during lactation.
In summary, this thesis has identified distinctions in the spontaneous activity patterns of TIDA neurons between male and female rats, with significant adaptations occurring during lactation. Moreover, these lactational adaptations showed a complete but gradual restoration to pre-lactational features following separation from pups. The observed variations in TIDA neuronal activity patterns based on sex and reproductive state align with known differences in the dopaminergic output of these neurons and the corresponding circulatory levels of PRL. Furthermore, this thesis provides further evidence supporting the involvement of Cx36 gap junctions and specific forms of chemical synaptic transmission in shaping TIDA neuron activity patterns. Based on these findings, it is reasonable to suggest that TIDA neuronal activity patterns play a crucial role in influencing the dopaminergic output of TIDA neurons and, consequently, PRL release.