Early experience modifies the postnatal assembly of autonomic emotional motor circuits in rats

Abstract

Rat pups that are repeatedly handled and separated from their dam exhibit altered adult behavioral, endocrine, and autonomic responses to stress, but the extent to which early handling and/or maternal separation (H/S) alters the development of circuits that underlie these responses is unknown. The present study tested the hypothesis that early H/S alters the postnatal assembly of synapses within preautonomic emotional motor circuits. Circuit development was traced by synapse-dependent retrograde transneuronal transport of pseudorabies virus (PRV) from the stomach wall. Control and H/S rats were analyzed between postnatal day 6 (P6) and P10, a period of rapid synaptic assembly among preautonomic circuit components. Pups in H/S groups were removed from their dam daily for either 15 min or 3 h beginning on P1, and were injected with virus on P8 and perfused on P10. Quantitative analyses of primary and transsynaptic PRV immunolabeling confirmed an age-dependent assembly of hypothalamic, limbic, and cortical inputs to autonomic nuclei. Circuit assembly was significantly altered in H/S pups, in which fewer neurons in the central amygdala, the bed nucleus of the stria terminalis, and visceral cortices were infected compared with age-matched controls. In contrast, H/S did not alter the assembly of paraventricular hypothalamic inputs to gastric autonomic neurons. H/S-related reductions in limbic and cortical transneuronal infection were similar in pups exposed daily to 15 min or 3 h maternal separation. These findings support the view that environmental events during early postnatal life can influence the formation of neural circuits that provide limbic and cortical control over autonomic emotional motor output.

Figure 1.

The routes of viral transport and regions subjected to quantitative analysis of the number of transneuronally infected neurons are illustrated. Previous studies in neonates and adult rats have demonstrated that injection of PRV-Bartha into the ventral wall of the stomach results in reproducible retrograde transneuronal infection of CNS neurons. Retrograde infection through the vagus nerve produces a first-order infection of parasympathetic vagal motor neurons within the DMV (D; blue). Replication and retrograde transneuronal passage of virus leads to the sequential infection of synaptically linked neurons in the adjacent NST (D; green) and AP (D; red). At longer survival intervals, virus passes transneuronally from the DVC to infect neurons in higher regions of the neuraxis. The present analysis focused on the hypothalamic PVN (C), the CeA (C), the BNST (B), and visceral cortices [insular cortex (IC) and medial prefrontal cortex (mPFC) (prelimbic and infralimbic)] (A). These areas are highlighted in red in the sagittal and coronal schematics, modified from Swanson (1998).

Figure 2.

Immunocytochemical localization of PRV-positive neurons in control NH/NS rat pups injected with virus on P8 and killed after 63-66 h, on P10. The magnitude of forebrain infection (PVN, BNST, CeA) in individual cases correlated with the extent of infection in the AP (Table 2). Forebrain infection was sparse in cases that exhibited robust infection of the left DMV and NST but only scattered infection of AP (A-D). In contrast, extensive infection of neurons in the PVN, BNST, and CeA invariably accompanied extensive AP infection (E-H). Scale bar: (in H) A-H, 100 μm. See Figure 1 for regional schematics.

Figure 3.

Inclusion criteria for the quantitative analysis of transneuronal infection of forebrain cell groups were based on the extent of infection in the AP. The borders of the three areas composing the DVC (i.e., DMV, NST, and AP) are demarcated (A) (see also Fig. 1). Rat pups included in quantitative analyses exhibited dense bilateral infection of the DVC, including a minimum of 500 infected AP neurons. A-D, Typical DVC infection in representative cases from each of the four experimental groups. Scale bar: (in D) A-D, 100 μm. See Figure 1 for regional schematics.

Figure 4.

Age-dependent assembly of preautonomic circuits. Large numbers of transneuronally infected neurons are evident in the PVN (D-F), BNST (J-L), and CeA (P-R) in control NH/NS rat pups injected with virus on P8 and killed on P10; evidence that these synaptic inputs from neurons in these regions to gastric autonomic output neurons are well established by this time. In contrast, significantly fewer infected neurons are evident within the PVN (A-C), BNST (G-I), or CeA (M-O) in NH/NS rat pups injected with virus on P4 and killed on P6, despite comparable infection within the hindbrain DVC in all cases (see also Fig. 3). Each row of photomicrographs depicts sections through rostral (left), intermediate (middle), and caudal (right) levels through each region. Scale bar: (in R) A-R, 100 μm. See Figure 1 for regional schematics.

Figure 5.

Bar graphs illustrating transneuronal infection of PVN neurons (normalized to AP infection within each case) in control NH/NS rat pups injected with virus on P4 or P8 and killed on P6 or P10, respectively, and in 15 min and 3 h H/S rat pups injected with virus on P8 and killed on P10 (group mean ± SE). Bars represent labeling within four parvocellular PVN subnuclei, the anterior parvocellular (PVNap), medial parvocellular (PVNmp), dorsal parvocellular (PVNdp), and posterior parvocellular (PVNpp). The final set of bars represents total PVN (PVN tot). Within each subregion, bars with different letters (i.e., A, B, or C) are significantly different (p < 0.05). Statistical comparisons were performed on log-transformed data that met assumptions of normality and homoscedasticity (see Results).

Figure 6.

Bar graphs illustrating transneuronal infection of CeA [medial (CeA m) and lateral (CeA l) subnuclei] and BNST neurons (normalized to AP infection within each case) in control NH/NS rat pups injected with virus on P4 or P8 and killed on P6 or P10, respectively, and in 15 min and 3 h H/S rat pups injected with virus on P8 and killed on P10 (group mean ± SE). Within each region, bars with different letters (i.e., A, B, or C) are significantly different (p < 0.05). Statistical comparisons were performed on log-transformed data that met assumptions of normality and homoscedasticity (see Results).

Figure 7.

Bar graphs illustrating transneuronal infection of neurons within the IN and IL/PL medial prefrontal cortices (normalized to AP infection within each case) in control NH/NS rat pups injected with virus on P4 or P8 and killed on P6 or P10, respectively, and in 15 min and 3 h H/S rat pups injected with virus on P8 and killed on P10 (group mean ± SE). Within each region, bars with different letters (i.e., A, B, or C) are significantly different (p < 0.05). Statistical comparisons were performed on log-transformed data that met assumptions of normality and homoscedasticity (see Results).

Figure 8.

The effect of daily repeated postnatal H/S on transneuronal infection of the PVN, BNST, and CeA is illustrated. Control NH/NS rat pups injected with virus on P8 and killed on P10 exhibited robust infection within each forebrain region (A-C). There was no significant effect of treatment on PVN transneuronal infection (A, D, G). In contrast, the BNST (B, E, H) and CeA (C, F, I) exhibited significantly reduced transneuronal PRV labeling in 15 min and 3 h H/S rat pups. Scale bar: (in I)A-I, 100 μm. See Figure 1 for regional schematics.