SUMMARY A critical feature of neural networks is that they balance excitation and inhibition to prevent pathological dysfunction. How this is achieved is largely unknown, though deficits in the balance contribute to many neurological disorders. We show here that a microRNA (miR-101) is a key orchestrator of this essential feature, shaping the developing network to constrain excitation in the adult. Transient early blockade of miR-101 induces long-lasting hyper-excitability and persistent memory deficits. Using target-site blockers in vivo, we identify multiple developmental programs regulated in parallel by miR-101 to achieve balanced networks. Repression of one target, NKCC1, initiates the switch in GABA signaling, limits early spontaneous activity, and constrains dendritic growth. Kif1a and Ank2 are targeted to prevent excessive synapse formation. Simultaneous de-repression of these three targets completely phenocopies major dysfunctions produced by miR-101 blockade. Our results provide new mechanistic insight into brain development and suggest novel candidates for therapeutic intervention.

Developing novel therapeutics for bipolar disorder (BD) has been hampered by limited mechanistic knowledge how sufferers switch between mania and depression-how the same brain can switch between extreme states-described as the "holy grail" of BD research. Strong evidence implicates seasonally-induced switching between states, with mania associated with summer-onset, depression with winter-onset. Determining mechanisms of and sensitivity to such switching is required. C57BL/6J and dopamine transporter hypomorphic (DAT-HY 50% expression) mice performed a battery of psychiatry-relevant behavioral tasks following 2-week housing in chambers under seasonally relevant photoperiod extremes. Summer-like and winter-like photoperiod exposure induced mania-relevant and depression-relevant behaviors respectively in mice. This behavioral switch paralleled neurotransmitter switching from dopamine to somatostatin in hypothalamic neurons (receiving direct input from the photoperiod-processing center, the suprachiasmatic nucleus). Mice with reduced DAT expression exhibited hypersensitivity to these summer-like and winter-like photoperiods, including more extreme mania-relevant (including reward sensitivity during reinforcement learning), and depression-relevant (including punishment-sensitivity and loss-sensitivity during reinforcement learning) behaviors. DAT mRNA levels switched in wildtype littermate mice across photoperiods, an effect not replicated in DAT hypomorphic mice. This inability to adjust DAT levels to match photoperiod-induced neurotransmitter switching as a homeostatic control likely contributes to the susceptibility of DAT hypormophic mice to these switching photoperiods. These data reveal the potential contribution of photoperiod-induced neuroplasticity within an identified circuit of the hypothalamus, linked with reduced DAT function, underlying switching between states in BD. Further investigations of the circuit will likely identify novel therapeutic targets to block switching between states.

Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native state. However, the applicability of cryofixation is limited by its incompatibility with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here, we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing for quality ultrastructural preservation for diverse EM applications.

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for methamphetamine abuse in individuals with HIV.

26Electron microscopy (EM) offers unparalleled power to study cell substructures at the 27 nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as 28 it captures cellular structures instantaneously in their near-native states. However, the 29 applicability of cryofixation is limited by its incompatibilities with diaminobenzidine labeling using 30 genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning 31 electron microscopy (SBEM). In addition, it is challenging to perform correlated light and 32 electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods 33 cannot be applied to address questions requiring optimal morphological preservation and high 34 temporal resolution. Here we developed an approach that overcomes these limitations; it 35 enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. 36Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory 37 organ and mouse brain. This optimization exploits the potential of cryofixation, allowing quality 38 ultrastructural preservation for diverse EM applications. 39Cryofixation is especially critical, and often necessary, for properly fixing tissues with cell 55 walls or cuticles that are impermeable to chemical fixatives, such as samples from yeast, plant, 56 Winey et al., 58 1995). As cryofixation instantaneously halts all cellular processes, it also provides the temporal 59 control needed to capture fleeting biological events in a dynamic process (Hess et al., 2000; 60 Watanabe et al., 2013; Watanabe et al., 2013;Watanabe et al., 2014). 61Despite the clear benefits of cryofixation, it is incompatible with diaminobenzidine (DAB) 62 labeling reactions by genetic EM tags. For example, APEX2 (enhanced ascorbate peroxidase) 63is an engineered peroxidase that catalyzes DAB reaction to render target structures electron 64 dense (Lam et al., 2015; Martell et al., 2012). Despite the successful applications of APEX2 to three-dimensional (3D) EM (Joesch et al., 2016), there has been no demonstration that APEX2 66 or other genetic EM tags can be activated following cryofixation. Conventionally, cryofixation is 67 followed by freeze-substitution (Steinbrecht and Müller, 1987), during which water in the sample 68 is replaced by organic solvents. However, the resulting dehydrated environment is incompatible 69 with the aqueous enzymatic reactions required for DAB labeling by genetic EM tags. 70 EM structures can also be genetically labeled with fluorescent markers through 71 correlated light and electron microscopy (CLEM). Yet, performing CLEM with cryofixed samples 72 also presents challenges. Fluorescence microscopy commonly takes place either before 73 cryofixation (Brown et al., 2009; Kolotuev et al., 2010;McDonald, 2009) or after the sample is 74 embedded (Kukulski et al., 2011;Nixon et al., 2009;Schwarz and Humbel, 2009). However, if 75 the specimen is dissected from live animals, the ti...