Periodic Reporting for period 1 - REDIRECT (Resolving sEx DIffeREnces in proCessing sensory informaTion)
Okres sprawozdawczy: 2023-06-01 do 2025-11-30
Mental disorders are associated with abnormal sensory information processing (SIP) at preadolescence, especially in males. Aberrant SIP is a neutral cross-disorder trait that can be translated between humans and animals and assessed as the inability to prevent/gate an overload of irrelevant information. The mechanisms underlying physiological and pathological SIP variability are unexplored. Particularly, the causative role of hyperdopaminergia, key to SIP deficits, has never been tested by isolating, spatially and temporally, this single neural component. How does this trait evolve into a mental disorder? Why does it occur more often in males?
REDIRECT will capitalize on our rodent model of prenatal cannabis exposure (PCE), which is the first of its kind to offer, at preadolescence, a sex bias in susceptibility to SIP deficits upon acute exposure to stress or cannabis. This animal model is amenable to molecular interrogation to probe causality for those resilience-promoting factors preventing females from developing a hyperdopaminergia and SIP deficits.
By combining viral strategies to trace and probe cell-type specific input-output reorganization of dopamine circuits (Aim 1), with single-cell transcriptome analyses (Aim 2), REDIRECT will decipher how female sex imposes distinct molecular changes to neurons and circuits, which are key to prevent the etiopathogenesis of SIP disorders. By testing selected candidate genes to probe causality for how sex modulates genes, developmental trajectories, and behaviour, we will decode (Aim 3) how discrete dopaminergic circuits can be reprogrammed sex-dependently.
REDIRECT will causally disentangle how sex differently affects genes, circuits, and behaviour to inform novel sex-specific and age-tailored therapies for SIP deficits, a cross-disorder trait typical of common and severe mental illnesses, including autism, attention deficit hyperactivity disorder, obsessive-compulsive disorder and schizophrenia.
REDIRECT will capitalize on our rodent model of prenatal cannabis exposure (PCE), which is the first of its kind to offer, at preadolescence, a sex bias in susceptibility to SIP deficits upon acute exposure to stress or cannabis. This animal model is amenable to molecular interrogation to probe causality for those resilience-promoting factors preventing females from developing a hyperdopaminergia and SIP deficits.
By combining viral strategies to trace and probe cell-type specific input-output reorganization of dopamine circuits (Aim 1), with single-cell transcriptome analyses (Aim 2), REDIRECT will decipher how female sex imposes distinct molecular changes to neurons and circuits, which are key to prevent the etiopathogenesis of SIP disorders. By testing selected candidate genes to probe causality for how sex modulates genes, developmental trajectories, and behaviour, we will decode (Aim 3) how discrete dopaminergic circuits can be reprogrammed sex-dependently.
REDIRECT will causally disentangle how sex differently affects genes, circuits, and behaviour to inform novel sex-specific and age-tailored therapies for SIP deficits, a cross-disorder trait typical of common and severe mental illnesses, including autism, attention deficit hyperactivity disorder, obsessive-compulsive disorder and schizophrenia.
During the first trimester of the project, I worked together with the Host Institution (HI) and the other team members to outline the resources we needed, in terms of animal facility space, laboratory configuration and equipment, and manpower. The main deliverables of the first trimester were to purchase the new patch-clamp set up and the cages to perform pre-pulse inhibition experiments, to extend the animal license for the use of TH::Cre rats and to buy the viral vectors (WP 0). We successfully obtained the ethical approval of the experiments to accomplish WP1-3 (aut. N. 218/2023-PR).
Since the beginning of the 4th trimester, we, in a parallel way, started to 1) probe cell-type specific input-output reorganization of dopamine circuits (WP1-2); while 2) providing the proof of principle of REDIRECT. We have so far carried out the analysis of three out of six planned nuclei (WP1-2). We used viral strategies, combined with electrophysiological analyses and optogenetic manipulations to perform a multimodal characterization of the inhibitory monosynaptic inputs to decipher the relative contribution of these inputs in the sex-specific effects of PCE. Preliminary analyses (WP 8) suggests remarkable circuit- and sex- specific dysfunction of two out of the three afferent terminals so far examined. PCE induces a circuit-specific deregulation of inhibitory afferents that differs between sexes.
As planned, by the end of 3rd trimester, we obtained the ethical approval (Aut. N. 14/2024-PR) to begin Aim 2 (WP 4-5), and next we obtained the plasmid for the RiboTag experiment (WP 4). We obtained the pAAV-DIO-RPL22HA-IRES-YFP from prof. McKnight, whom developed the ribosomal tagging strategy to analyze mRNA translation and gene expression in a cell-specific manner in complex tissues. This was packaged in a Cre-dependent retrograde viral vector in an Italian facility at the International Centre for Genetic Engineering and Biotechnology. This retrograde strategy will enable us to halve the number of bilateral stereotactic surgeries to be performed at PND7 during year 3.
The implementation of WP1-2 and 4 has been subjected to a delay, due to unexpected biological issues related to the fertility of the transgenic rat colony (TH::Cre). This forced us to almost halt the experimentation during the 7th trimester. Hence, I purchased a second batch of transgenic animals that only arrived at the end of this period (month 22). I plan to complete WP 4 and 5 by the beginning of month 30 and 32, respectively.
Since the beginning of the 4th trimester, we, in a parallel way, started to 1) probe cell-type specific input-output reorganization of dopamine circuits (WP1-2); while 2) providing the proof of principle of REDIRECT. We have so far carried out the analysis of three out of six planned nuclei (WP1-2). We used viral strategies, combined with electrophysiological analyses and optogenetic manipulations to perform a multimodal characterization of the inhibitory monosynaptic inputs to decipher the relative contribution of these inputs in the sex-specific effects of PCE. Preliminary analyses (WP 8) suggests remarkable circuit- and sex- specific dysfunction of two out of the three afferent terminals so far examined. PCE induces a circuit-specific deregulation of inhibitory afferents that differs between sexes.
As planned, by the end of 3rd trimester, we obtained the ethical approval (Aut. N. 14/2024-PR) to begin Aim 2 (WP 4-5), and next we obtained the plasmid for the RiboTag experiment (WP 4). We obtained the pAAV-DIO-RPL22HA-IRES-YFP from prof. McKnight, whom developed the ribosomal tagging strategy to analyze mRNA translation and gene expression in a cell-specific manner in complex tissues. This was packaged in a Cre-dependent retrograde viral vector in an Italian facility at the International Centre for Genetic Engineering and Biotechnology. This retrograde strategy will enable us to halve the number of bilateral stereotactic surgeries to be performed at PND7 during year 3.
The implementation of WP1-2 and 4 has been subjected to a delay, due to unexpected biological issues related to the fertility of the transgenic rat colony (TH::Cre). This forced us to almost halt the experimentation during the 7th trimester. Hence, I purchased a second batch of transgenic animals that only arrived at the end of this period (month 22). I plan to complete WP 4 and 5 by the beginning of month 30 and 32, respectively.
As proof-of-principle of REDIRECT hypothesis, we demonstrated the essential role of dopamine in the detrimental effects of acute stressors on sensory information processing, as indexed by deficits in sensorimotor gating functions, in preadolescent rats. We showed the causal link between dopamine signaling and the engagement of stress axis for the individual ability to inhibit behavioral responses to incoming sensory information. As inter-individual variability is normally distributed, and REDIRECT leverages an animal model where the individuals represent the tail ends of such a distribution (vulnerable vs resilient), we were able to prove that enhancing dopamine neuron activity is required for inducing vulnerability to the deteriorating effects of acute inescapable stressors on sensorimotor gating functions. We used a chemogenetic approach to normalize or enhance dopamine signaling in vulnerable (PCE males) and resilient (PCE females) subjects, respectively, and showed that the male-specific susceptibility to stress-induced psychotic-like phenotype induced by PCE depends on a hyperdopaminergic phenotype.
We believe that these findings represents a substantial advance in knowledge because PCE children exhibit proneness to psychotic-like experiences and some psychopathological phenotypes where males face a higher risk.
We believe that these findings represents a substantial advance in knowledge because PCE children exhibit proneness to psychotic-like experiences and some psychopathological phenotypes where males face a higher risk.