Results we have previously reported [31,32,44], but also in a
A .E. Pekary et al. Brain Research 884 2000 174 –183
177
2.8. Statistical analysis shown. Post-ECS hindlimb paralysis reduced the number
of ECS animals. This was most likely due to the use of Statistical comparisons were made with the aid of
65-day-old, 320 g body weight, instead of 55-day-old, 250 Statview Abacus Concepts, Inc., Berkeley, CA, a statisti-
g animals. The three rats that received both ECS and a cal software package for the Macintosh computer. All
swim score were not sufficient for meaningful correlation multigroup comparisons were carried out by one way
of swimming behavior with levels of TRH and TRH- analysis of variance using post-hoc Scheffe contrasts with
related peptides in various brain regions. the control group.
Nevertheless, highly significant increases in the levels of Ps4–IR, TRH–IR, and TRH–Gly–IR were observed in a
variety of brain regions, not only in the limbic system as
3. Results we have previously reported [31,32,44], but also in a
number of non-limbic areas as summarized in Table 1. The combined use of HPLC and EEP RIA see Fig. 1
Consistent with our previous reports [31,32,44], ECS for representative EEP–IR profiles following HPLC of
increased Ps4–IR, TRH–IR and TRH–Gly–IR levels in
2
pooled tissue extracts demonstrated that EEP, TRH, Tyr – pyriform cortex, hippocampus, entorhinal cortex, and
2
TRH, and Phe –TRH all occur in rat brain but their amygdala. TRH–Gly–IR levels in the amygdala were at
relative concentrations vary markedly between specific least 10-fold higher than the corresponding levels in any
brain regions. A minor EEP–IR peak with a retention time other brain region studied. In addition, we found that ECS
of about 15 min has not yet been identified. It is apparent increased Ps4–IR levels in hypothalamus, posterior cingu-
that in lateral cerebellum and striatum Fig. 1 and in late and lateral cerebellum and raised the TRH–Gly–IR
amygdala, anterior cingulate, entorhinal cortex, and frontal concentration in the hypothalamus.
cortex results not shown, the EEP–IR and TRH–IR The combined effect of ECS and swimming was to
consists of a mixture of two or more different TRH-like decrease the level of TRH–Gly–IR in the anterior cingu-
2
peptides. Following ECS the peak corresponding to Phe – late Fig. 2, an observation that has recently been
TRH in lateral cerebellum increased six-fold results not confirmed in a similar protocol using vagal nerve stimula-
shown. We have not been successful in our attempts to tion instead of ECS S. Krahl, A.E. Pekary, A. Sattin,
2
produce more specific antibodies to EEP, Tyr –TRH and unpublished results. This result is consistent with in-
2
Phe –TRH using currently available methodologies [28]. creased conversion of TRH–Gly–IR for anterior cingu-
2 2
2
This is the first report of Tyr –TRH and Phe –TRH late, a mixture of EEP–Gly and Tyr –TRH–Gly to EEP
2
occurring in mammalian brain. and Tyr –TRH, respectively, the major EEP–IR peaks
Three ECS administered on consecutive days with found after HPLC and EEP RIA of this tissue result not
progressively increasing current, with or without forced shown, and release of these peptides [4,30]. This com-
swimming, did not affect total body weights or tissue bined suppressive effect of ECS and forced swimming on
weights in the brain regions that were dissected results not TRH–Gly–IR concentration in the anterior cingulate is in
Fig. 1. Profile of EEP immunoreactivity EEP–IR following HPLC fractionation of striatum and lateral cerebellum. Injected samples were prepared from pooled extracts n56 of left panel post-ECS striatum three swim plus three non-swim with the standard acetonitrile gradient superimposed; right
panel corresponding profile for sham-ECS lateral cerebellum six non-swim.
178 A
Table 1 Effect of electroconvulsive seizure ECS; three swim plus three non-swim versus sham ECS control; six swim plus six non-swim treatment on levels of
Ps4 immunoreactivity Ps4–IR; TRH–IR, and TRH–Gly–IR, ng g wet weight, levels in various brain regions of male Wistar rats, 300 to 350 g body
a
weight Brain region
Pyriform cortex Hypothalamus
Hippocampus Entorhinal cortex
Amygdala Posterior cingulate
Lateral cerebellum TRH–IR
ECS 6 0.960.3
27.964.3 2.360.3
1.460.2 3.660.7
0.4260.14 0.2660.04
Control 12 0.460.2
25.664.9 1.060.3
1.060.4 2.660.4
0.4560.15 0.3060.08
t 4.97
0.96 8.87
2.4 3.46
0.48 1.08
P ,0.001
,N.S. ,0.001
,0.05 ,0.005
N.S. N.S.
TRH–Gly–IR ECS 6
32.4610.9 19.069.9
18.066.5 28.6615.1
3226172 32616
10.364.9 Control 12
19.7611.0 10.066.1
7.263.0 15.467.2
133682 30613
8.164.1 t
2.30 2.40
4.92 2.55
3.12 0.27
1.02 P
,0.05 ,0.05
,0.001 ,0.05
,0.01 N.S.
N.S. Ps4–IR
ECS 6 7.263.7
33.6616.5 4.862.5
5.862.7 10.663.4
5.462.9 2.560.7
Control 12 3.362.7
16.368.2 1.360.8
2.761.4 4.462.2
1.760.9 1.060.8
t 2.51
3.01 4.69
3.25 4.71
4.21 4.11
P ,0.05
,0.01 ,0.001
,0.01 ,0.001
,0.001 ,0.001
a
P-values are for the two-tailed Student’s t-test.
marked contrast with the observed increase in TRH–Gly– IR, TRH–IR and Ps4–IR levels in other brain regions,
summarized in Figs. 3–5. TRH, TRH–Gly and Ps4 are all prepro-TRH-derived
2
peptides. The TRH-like peptides, EEP, Tyr –TRH, and
2
Phe –TRH are the products of other, as yet uncharacter- ized, precursor proteins [31]. Nevertheless, expression of
these peptides appears to vary in a similar manner to the prepro-TRH-derived peptides as evidenced by the signifi-
cant correlation of EEP–IR and Ps4–IR levels in a variety of brain regions as displayed in Figs. 6 and 7.